The Methylerythritol Phosphate Pathway to ... - ACS Publications

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The Methylerythritol Phosphate Pathway to Isoprenoids Annika Frank* and Michael Groll* Center for Integrated Protein Science Munich (CIPSM) at the Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany ABSTRACT: Isoprenoids constitute one of the most diverse classes of natural products. As a compound class, they are essential to basic metabolic processes including cell-wall biosynthesis, post-translational protein modifications, and signaling. In addition, isoprenoid secondary metabolites are highly valuable natural products with a wide range of biotechnological applications. The biosynthesis of their two universal building blocks, isopentenyl diphosphate and dimethylallyl diphosphate, was thought to proceed exclusively by way of mevalonate as a key intermediate until a novel pathway involving methylerithritol phosphate (MEP) was discovered in the early 1990s. In this review, we describe the seven enzymes of the MEP pathway, along with their discoveries, threedimensional structures, and mechanisms. The latter include examples of remarkable enzyme catalysis including an unusual cytidilation reaction and the use of iron−sulfur cluster cofactors in reductive ring opening and hydroxy-group elimination. Furthermore, isoprenoid biosynthesis shows a characteristic species distribution. A brief overview highlights the MEP pathway’s potential as a selective drug target, which is absent in humans but essential to the survival of many important bacterial and apicomplexan pathogens.

CONTENTS 1. Introduction 2. The Methylerythritol Phosphate Pathway 2.1. Discovery 2.2. DXS 2.2.1. Discovery 2.2.2. Structure 2.2.3. Mechanism 2.3. IspC 2.3.1. Discovery 2.3.2. Structure 2.3.3. Mechanism 2.4. IspD 2.4.1. Discovery 2.4.2. Structure 2.4.3. Mechanism 2.5. IspE 2.5.1. Discovery 2.5.2. Structure 2.5.3. Mechanism 2.6. IspF 2.6.1. Discovery 2.6.2. Structure 2.6.3. Mechanism 2.6.4. Bifunctional IspDF Enzymes 2.7. IspG 2.7.1. Discovery 2.7.2. Structure 2.7.3. Mechanism 2.8. IspH 2.8.1. Discovery 2.8.2. Structure 2.8.3. Mechanism © 2016 American Chemical Society

3. Drug Targets in the Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis 4. Conclusions and Outlook Author Information Corresponding Authors ORCID Author Contributions Notes Biographies Acknowledgments Abbreviations References

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1. INTRODUCTION With over 55000 molecules, isoprenoids constitute one of the largest classes of natural compounds known to date.1 All members of this group, no matter how complex, share a common five-carbon isoprene building block. This basic unit can be linked by a head-to-tail (regular) or head-to-head (irregular) condensation reaction, following the so-called “isoprene rule”.2 This extension, along with the introduction of functional groups such as aldehydes, alcohols, ketones, peroxides, ethers, esters, and carboxylic esters, leads to a remarkable diversity of achiral or chiral, linear, and cyclic molecules.3−5 These include members of the well-characterized mono-, sesqui-, di-, and triterpene families, which have been reviewed extensively.6−11 Once modified, the isoprene units become isoprenoids or, as they are also called, terpenoids.3 Special Issue: Unusual Enzymology in Natural Products Synthesis Received: August 11, 2016 Published: December 20, 2016 5675

DOI: 10.1021/acs.chemrev.6b00537 Chem. Rev. 2017, 117, 5675−5703

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Figure 1. Isoprenoid biosynthetic pathways. Isoprenoids constitute one of the major classes of natural compounds and are essential metabolites in all kingdoms of life. Their biosynthesis occurs by two distinct pathways named after their key intermediates: MEP (left) and MVA (right). The MVA pathway is found in higher, complex organisms including animals and fungi, as well as in archaea and some bacteria. Most other prokaryotes and the eukaryotic apicomplexa, including many major pathogens, make use of the MEP pathway. Plants show compartmentalized isoprenoid biosynthesis in their cytosol (MVA) and plastids (MEP). Shunt pathways, such as production of isopentenyl phosphate, have also been described.

inside and outside the cell. This includes their function as hormones in many higher eukaryotes.3,4 Even though plant isoprenoids in particular have been in use as medicinal, flavor, and aroma compounds for millennia, little was known about their chemical structures and properties until the beginning of the 19th century. This changed with Ruzicka’s formulation of the isoprene rule.2 Between the 1940s and 1960s, the structure elucidations of cholesterol and other natural products led to the identification and characterization of

Isoprenoids are essential for a variety of ubiquitous cellular processes, including transcription and post-translational modifications, cell-wall biosynthesis, electron transport, photosynthesis, intracellular signaling, secreted defense mechanisms, and protein degradation. They are found in all kingdoms of life, with a large subgroup comprising secondary metabolites produced by microorganisms, fungi, and plants.8,12 Their physical properties, including low molecular weights and high vapor pressures, render them important messenger molecules 5676


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natural products. The MVA pathway has since been discovered in the cytosolic fraction of plants, fungi, and animals, as well as in a few bacteria. Deviations from the classical enzyme cascade include an alternative route to IPP catalyzed by a phosphomevalonate decarboxylase and isopentenyl phosphate kinase, which serves as an exclusive source of IPP in archaea and complements the full MVA pathway in plants.27−29 In addition to such partial modifications, however, isotopicexchange experiments in different organisms hinted at further inconsistencies with the entire reaction sequence.30−32 In the 1990s, these were analyzed in more detail by the research groups of Rohmer and Arigoni and led to the proposition of a completely new biosynthetic pathway for IPP and DMAPP. The incorporation of labeled precursors, 13C-glucose, pyruvate, and acetate, among others, led to the identification of two alternative starting molecules. A C2 unit derived from decarboxylation of pyruvate was proposed to condense onto a C3 unit, glyceraldehyde-3-phosphate (GAP), to form 1deoxyxylulose 5-phosphate (DXP). As C1, C2, and C4 of IPP were shown to derive from the C3 unit whereas C3 and C5 originated from the 2-carbon precursor, a subsequent transposition was suggested to yield the final isoprenoid building block IPP.33−36 In the following years, this simple “transposition” has turned out to be a complex sequence of reductions, transfer reactions, phosphorylations, and cyclizations, followed by iron−sulfur-cluster-mediated ring opening and further reductions. All seven enzymes responsible have since been characterized with respect to their structures and functions, as well as their often unusual and unprecedented reaction mechanisms. Together, they catalyze what is now known as the nonmevalonate, DXP, or methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis (Figure 1, left). Whereas MVA-based biosynthesis exclusively provides IPP and DMAPP building blocks in animals, fungi, archaea, and a few bacteria, the MEP pathway is the sole source of isoprenoids in most other bacteria, as well as the parasitic apicomplexa. The latter two groups include important human pathogens such as Helicobacter, Salmonella, and Chlamydia as well as Plasmodium and Toxoplasma species. A few bacteria exhibit both pathways and use them complementarily, whereas the genomes of others, such as Mycoplasma species, encode neither and their parasitic forms rely on their host cells for isoprenoid deliveries. In plants, biosynthesis is compartmentalized, with the MVA and MEP enzymes being localized in the cytosol and plastids, respectively. Whereas each of the pathways supplies the building blocks for a distinct set of downstream terpenoids, metabolic crosstalk takes place between them.37,38 This unique distribution makes isoprenoid biosynthesis and, in particular, the enzymes of the MEP pathway highly attractive drug targets. The absence of homologous proteins in humans and other animals enhances selectivity and prevents undesired side effects of anti-infectives. At the same time, the abundance of structural and mechanistic data facilitates inhibitor design. This also applies to the development of herbicides, as inhibition of plastid isoprenoid biosynthesis is detrimental to plant growth and cannot be compensated by the cytosolic MVA pathway.38 In addition to its mere target function, the MEP pathway is also interesting from a biocatalytic perspective, for example, in the production of aroma and flavor compounds or the synthesis of plant-derived biofuels.39 In summary, isoprenoid biosynthesis by the methylerythritol phosphate pathway consists of a series of unusual enzymatic

their basic isoprenoid building blocks, namely, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).2,13−15 Experiments with isotopically labeled acetate showed that these fundamental units are derived from acetylCoA, and several research groups subsequently identified and characterized a corresponding biosynthetic reaction sequence.13 This was termed the mevalonate pathway, after its key intermediate (3R)-3,5-dihydroxy-3-methylpentanoic acid (mevalonic acid, MVA) (Figure 1). The mevalonate biosynthetic pathway is initiated by two transferase reactions, each involving the loss of a coenzyme A (CoA) moiety. It was found that the Claisen condensation of two molecules of acetyl-CoA is catalyzed by an acetoacetyl-CoA thiolase16−18 and followed by a synthase-mediated aldol reaction between the β-ketothioester and an additional acetylCoA molecule. This sequence yields the first six-carbon intermediate, (S)-3-hydroxy-3-methylglutaryl-CoA (HMGCoA). Both enzyme catalysts form covalent adducts with their respective reaction intermediates and are cofactorindependent. HMG-CoA synthase, however, requires a water molecule for hydrolytic product release.17,19−21 A two-step, reduced nicotinamide adenine dinucleotide phosphate(NADPH-) dependent reduction subsequently leads to the formation of MVA and the concomitant release of CoA. The reaction is catalyzed by HMG-CoA reductase (HMGR) and follows a characteristic ping-pong mechanism of sequential CoA release and formation of a mevaldehyde intermediate. Protonation of the aldehyde moiety eventually leads to the formation of (R)-mevalonate. The reaction of HMGR was shown to be irreversible and is targeted efficiently by cholesterol-lowering drugs of the statin family.17,22,23 The biosynthetic steps following mevalonate production involve two adenosine triphosphate- (ATP-) dependent phosphorylations at its terminal hydroxy group. These result in the formation of mevalonate-5-phosphate and mevalonate-5-diphosphate and are catalyzed by mevalonate kinase (MK) and phosphomevalonate kinase (PMK), respectively.17 Although ATP is used preferentially by MK, the nucleotide moiety is replaceable, and cytidine, guanosine, and uridine triphosphate show comparable activities in vitro. Adenosine diphosphate (ADP), on the other hand, is not a substrate.24 PMK completes the formation of the pyrophosphate moiety by transferring a second terminal phosphate, now acid-labile, from ATP. This reaction was shown to be easily reversible upon addition of ADP.15,25 To obtain the final isoprenoid product of the MVA pathway, dehydration and decarboxylation reactions are required. Both were suggested to occur through a concerted and, at the time of discovery, unprecedented mechanism catalyzed by mevalonate diphosphate decarboxylase (MDD). The consumption and release of stoichiometric amounts of ATP and inorganic phosphate, respectively, suggested phosphorylation of the C3hydroxy moiety, resulting in an improved leaving group at this position. Dephosphorylation would then create a carbocation intermediate, which would, in turn, provide a suitable electron sink for decarboxylation and formation of the terminal alkene of the isopentenyl diphosphate product.15,17 Early experiments on the biosynthesis of squalene had already indicated the incorporation of DMAPP, rather than IPP, into the final terpene structure. The conversion between the two isoprenoids was found to be catalyzed enzymatically by an isopentenyl pyrophosphate isomerase (IPI).26 This step represents the ultimate reaction leading from acetyl-CoA as a simple metabolic product to two of the most ubiquitous building blocks of 5677


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Figure 2. Timeline of discoveries in the MEP pathway for isoprenoid biosynthesis. Some of the major findings, starting from the elucidation of isoprenoid structures and the mevalonate pathway and including the discoveries of individual MEP catalysts, are shown. Selected key publications are referenced.

reactions, which jointly contribute to the efficient production of building blocks for tens of thousands of natural products. In this review, the discovery, structural, and mechanistic characterization of its key players are discussed, with a particular focus on some of the unique reactions that they catalyze. In addition, a brief overview of the pathway’s role as a highly selective drug target is provided, concluding with an outlook regarding some of the future prospects for research into this unusual and fascinating enzyme cascade.

2. THE METHYLERYTHRITOL PHOSPHATE PATHWAY 2.1. Discovery

The mevalonate pathway was widely accepted as the exclusive source of isoprenoid building blocks for many years following its discovery (Figure 2). Increasing inconsistencies were observed, however, in isotopic labeling experiments, showing a species-specific lack of incorporation of the presumed starting material and intermediates into various terpenoids. In the early 1960s, the absence of signals from [2-14C]-mevalonate in mycobacterial vitamin K12 as well as in β-carotene of the alga Chlorella pyrenoidosa was noted. A few years later, Ramasarma and co-workers observed that neither [1-14C]-acetate nor [2-14C]-mevalonate could be used to label coenzyme Q in bacteria, whereas for molds, it was successfully incorporated. Similar findings were later made for other terpenoids such as ubiquinones and bacterial hopanes.40−44 The possibility of alternative pathways to isoprenoids was raised as a consequence, but suggestions about its nature remained vague and controversial. A potential acetolactate pathway was quickly disproved, whereas other proposals strongly resembled the MVA-based biosynthesis or could not be substantiated experimentally.31,43 It was shown repeatedly, however, that acetyl CoA could not always serve as a universal IPP precursor and that alternative reactions and catalysts must therefore exist for the initial biosynthetic steps of the isoprenoids in question.31,42,43 Eventually, a completely novel pathway to the isopentenyl building blocks was independently discovered by the research groups of Rohmer and Arigoni in the early 1990s.34−36 While working on terpenoid production in bacterial species (including Escherichia coli and Zymomonas mobilis) and Gingko biloba plants, the two groups provided the first detailed descriptions of the starting molecules required for IPP biosynthesis and their metabolic origin. Feeding experiments with 13C-glucose allowed tracing of each of its six carbon atoms into IPP (Figure 3). A C2

Figure 3. Discovery of the methylerythritol phosphate pathway. Isotopic-exchange experiments revealed discrepancies with the classical route to isoprenoids and helped identify both starting molecules of the alternative pathway. 13C labeling allowed tracing of glucose carbon atoms into the universal precursors IPP and DMAPP at a ratio of 6:1 and established its origins in the C2 and C3 bodies GAP (solid circles) and pyruvate (open circles, one carbon lost by decarboxylation), respectively. Whereas pyruvate can be derived equally from glucose C1 to C3 (blue) or C4 to C6 (green), the three carbon atoms of GAP originate exclusively from C4 to C6 in organisms using the Entner− Doudoroff pathway (*). If glycolysis occurs by the Embden− Meyerhoff pathway, interconversion from DHAP ensures a balanced pool of GAP from glucose C1 to C3 or from glucose C4 to C6. One example for a possible isotope-labeling pattern is shown. The numbering corresponds to the numbering of the carbon atoms of glucose.

body of dual origin, most likely derived by decarboxylation of pyruvate and comprising either C2 and C3 or C5 and C6 of glucose, was shown to provide the isopentenyl C3 and C5. The remaining C1, C2, and C4 atoms of the five-carbon building block originated from a single triose incorporating C1−C3 or C4−C6 of glucose. This triose was subsequently identified as glyceraldehyde-3-phosphate (GAP).35 Variations in the labeling pattern of this C3 body between the various organisms analyzed were found to arise from differences in their respective 5678


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Figure 4. Three-dimensional structure of E. coli DXS [Protein Data Bank identifier (PDB ID) 2O1S]. (a) In its overall fold, the dimeric DXP synthase resembles transketolases and other members of the ThDP-dependent enzyme family. The three domains with their typical α/β fold are each in contact with their counterparts in the neighboring subunit. (b) ThDP is bound between domains I and II of each monomer and coordinates a Mg2+ ion. A close-up of the cofactor (inset) highlights solvent exposure of the catalytically active thiazolium moiety.

In a parallel approach, Rohmer and co-workers identified the same E. coli open reading frame through a proximity search around the ispA gene. 46 The latter encodes farnesyl diphosphate synthase, one of only two catalysts known at that point to be involved in downstream isoprenoid biosynthesis. Again, the overexpressed gene product was able to convert pyruvate and GAP into DXP. It was also shown to accept glyceraldehyde as a substrate to give the unphosphorylated deoxyxylulose as an additional, yet less favored, product. Sequence and homology analyses revealed the presence of a ThDP binding motif in addition to other interesting features, such as a conserved histidine residue involved in proton transfer in the related transketolases. Both groups also noticed the similarities between E. coli dxs and the cla1 gene in Arabidopsis thaliana, disruption of which is connected to impaired photosynthesis.47 This already provided an early indicator of the compartmentalization of isoprenoid biosynthesis in plants, which was analyzed in more detail in the following years. 2.2.2. Structure. To date, DXS structures have been determined from two bacterial species, E. coli and the extremophilic Deinococcus radiodurans.48 In accordance with their sequence similarities and related catalytic activities, the two enzymes closely resemble each other. Their basic functional units are characteristic of the ThDP-dependent enzymes (of which they form a subfamily) and particularly reminiscent of members of the functionally related transketolases (Figure 4a).49,50 Each monomer consists of three domains arranged in a linear, but slightly bent assembly, whose structural complexity decreases from the N- to the C-terminus. As a common feature, each of these domains shows a similar α/ β fold of a central, mainly parallel, five- or six-stranded β-sheet, sandwiched by surrounding α-helices. In solution, the DXS protein assembles into a closely associated dimer in which each domain is in direct contact with its counterpart in the neighboring monomer. This arrangement has a significant impact on the active-site composition, as the catalytic center consists exclusively of residues from the two most N-terminal domains (I and II) of a single monomer. In contrast, members of the transketolase family place their most N-terminal domain (I) on top of the central and C-terminal parts of the adjacent monomer (II and III, respectively) in a “twisted” conformation.

glycolytic pathways. In higher plants and microorganisms such as E. coli, the Embden−Meyerhoff pathway produces dihydroxyacetone phosphate (DHAP) and GAP. Both can be interconverted and lead to a balanced C3 precursor pool from C1−C3 or C4−C6 of glucose. Glycolysis in bacteria such as Z. mobilis, on the other hand, proceeds through the Entner− Doudoroff pathway and leads to the production of GAP exclusively from C4−C6 and of pyruvate from C1−C3 of glucose (which is incorporated as the C2 body after decarboxylation). This assignment of pyruvate and GAP as starting molecules in the newly discovered pathway enabled the identification of their condensation product, the unbranched 1-deoxyxylulose 5phosphate (DXP), as a first reaction intermediate.35 In subsequent years, the enzyme catalysts responsible for DXP formation and all downstream reactions leading to IPP and DMAPP formation were identified and meticulously characterized. Their discovery and properties, including many of their surprising and unusual structural and mechanistic features, are described in the following sections. 2.2. DXS

2.2.1. Discovery. Analogously to the pathway itself, the first MEP catalyst was discovered and characterized independently by two research groups. As the required chemical reaction had been identified previously as an acyloin condensation between pyruvate (after its decarboxylation to hydroxyethyl thiamin-PP) and GAP,34,36 the newly available E. coli genome could now be analyzed for the corresponding enzymatic activities. Sprenger and co-workers thus searched for reactions involving the decarboxylation of pyruvate.45 These are particularly wellknown for enzymes dependent on the cofactor thiamin diphosphate (ThDP) and include the transketolases, pyruvate decarboxylases, and the E1 component of the pyruvate dehydrogenase complex (PDHC). Their search revealed an open reading frame (ORF) whose protein product was able to accept pyruvate and GAP as substrates and convert them into DXP. Purification of the corresponding enzyme yielded a 65kDa protein with an activity that was entirely dependent on the presence of its ThDP cofactor. The analysis of related sequences in known bacterial and plant genomes subsequently led to the proposal of an entirely new, deoxyxylulose phosphate synthase (DXS) enzyme family. 5679


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Figure 5. DXS catalyzes the conversion of pyruvate and GAP into DXP. (a) The ThDP cofactor is activated by deprotonation and conversion into its dipolar ylide form. This may be mediated by catalytic acid/base residues in the active site or through intramolecular proton abstraction. (b) The enzymatic reaction corresponds to a formal acyloin condensation. Carbon−carbon bonds are formed by nucleophilic attack of the cofactor ylide on the pyruvate donor and of the activated aldehyde, formed after decarboxylation, on the GAP acceptor molecule. The carbanion intermediate is in equilibrium with its enamine form.

2.2.3. Mechanism. The reaction catalyzed by DXS corresponds to the well-characterized enzymatic α-hydroxyketone (acyloin) condensation and proceeds analogously to the mechanism observed in transketolases.45,46,50 Prior to substrate binding, activation of the thiamine diphosphate cofactor by deprotonation of the thiazolium ring’s C2 carbon is required (Figure 5a). This reaction is unfavorable in the isolated cofactor because of the high pKa of the proton but enabled by the chemical environment created inside the enzyme catalyst. Two possibilities exist for the identity of the catalytic base. The Vshaped conformation adopted by ThDP upon binding to the active site allows deprotonation of C2 by N4 of the (now adjacent) aminopyrimidine ring.53 Alternatively, a glutamate and an arginine side chain (Glu370 and Arg398 using the E. coli numbering) were proposed to play a role in this cofactor activation.48 Deprotonation converts the ThDP thiazolium ring into its carbanion ylide form and enables a nucleophilic attack on pyruvate as the first step of a sequential reaction mechanism (Figure 5b). The electrophilic iminium of the thiazole ring subsequently acts as an electron sink in substrate decarboxylation. The resulting carbanion/enamine, also known as an activated aldehyde, is able to carry out an “umpolung”-type nucleophilic attack on the carbonyl carbon of the GAP acceptor.53 A final deprotonation step releases the DXP product and regenerates the ThDP ylide for another round of catalysis. The identity of the catalytic acid/base involved in this last reaction remains to be determined. Based on mutagenesis studies and sequence alignments, a highly conserved histidine (His49 in E. coli) has been proposed to fulfill this function.51 Only a complex structure of DXS with one or both of its

As a result, both subunits contribute residues to the respective active sites. This distinction between an intra- or intermolecular composition of the catalytic center is characteristic for ThDPdependent enzymes and separates them into groups according to their domain sequence and linkage. Interestingly, cofactor binding and active-site architecture, including the identity of several key catalytic residues, are nonetheless highly conserved throughout the family.48,49 In the DXP monomer, ThDP is bound in a defined pocket at the interface between domains I and II (Figure 4b). It creates a direct connection between the C-termini of their respective βsheets and adopts a V-shaped conformation typical for the active form of the cofactor. In line with the chemical moieties with which they interact, domains I and II are thus also known as the pyrophosphate (PP) and (amino-)pyrimidine (PYR) domains, respectively. Whereas most of the prosthetic group is enclosed by the enzyme, the catalytically relevant thiazolium ring and, in particular, its C2 carbon are exposed to solvent and accessible for substrate binding in the PYR domain. The diphosphate, on the other hand, interacts with a variety of polar residues in the PP domain and participates in the coordination of a magnesium ion. Although both E. coli and D. radiodurans structures were determined in their holo (cofactor-bound) forms, ligand complexes with substrate, intermediates, and product remain elusive. The modeling of pyruvate and GAP substrates into the active site and mutagenesis studies have nonetheless identified a number of residues with potential functions in catalysis. These include a histidine suggested to act as a catalytic acid/base in the condensation reaction, a glutamate/arginine pair involved in cofactor activation, and two arginines interacting with the GAP substrate.48,49,51,52 5680


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Figure 6. Tertiary and quarternary structures of IspC. (a) Analogously to DXS, the IspC monomer can be subdivided into three domains: an Nterminal domain (blue) with the NADPH binding motif, a catalytic central domain (yellow/orange) with the substrate binding site and flexible loop region (orange), and a C-terminal domain consisting of four helix bundles (green). In solution, two monomers of IspC assemble into a homodimer by forming an extended β-sheet between the two central domains (PDB ID 1Q0Q). (b) A superposition between the apo (1), NADPH (2), and both NADPH- and DXP- (3) bound structures highlights the flexible loop movement involved in cofactor and substrate binding (PDB IDs 1Q0Q, 1K5H, 1JVS).

NADPH-dependent reduction to MEP. The reaction was shown to depend on the presence of divalent cations, with a preference for Mn2+, and to accept NADH as a cofactor with a 100-fold decrease in activity. YaeM, later renamed DXP reductoisomerase (DXR) or IspC, was thus identified as the second component of the novel MEP pathway.56 Unlike DXP, its metabolic reaction product is unique, and IspC catalysis is often described as the first committed step in isoprenoid biosynthesis. 2.3.2. Structure. The first apo structure of an IspC reductoisomerase from E. coli was published in 2002. It was immediately succeeded by the structure of a complex between the enzyme and its NADPH cofactor. Further structures with the catalytically important Mn2+ ion and the inhibitor fosmidomycin, which structurally mimics the substrate, and with NADPH and fosmidomycin followed. The first quarternary complex between IspC, NADPH, Mg2+, and fosmidomycin was released in 200758−62 (PDB IDs 1K5H, 1JVS, 1ONP, 1Q0H, and 2EGH). All enzymes, irrespective of their source organism, exhibit a typical architecture with a number of unusual structural features. IspC homologues were found to associate into homodimers in which two V-shaped monomers associate along their most apical β-sheets to form a saddle-like quarternary structure (Figure 6a). Their multimeric state, which differed between crystal structures and initial experimental results, was simultaneously confirmed by size-exclusion chromatography. Each monomer can be subdivided into three distinct domains: An N-terminal dinucleotide-binding domain harbors two Rossmann folds and acts as binding site for NADPH. In contrast to the classical motif of six parallel β-sheets, the IspC cofactor is anchored in a seven-stranded sheet, resulting from the insertion of an additional α/β element into the secondary structure.59 The central domain represents the catalytic center of the enzyme and is crucial for mediating conformational changes required for substrate binding and turnover. Its fold consists of two parallel open-faced β-sheets, followed by two antiparallel open-faced β-sheets. These are connected by loops

substrates, intermediates, or products, however, will help identify all residues required for catalysis. A few unique features distinguish the DXS reaction from those of other ThDP-dependent enzymes.52 It was proposed to follow a random sequential, preferred order mechanism involving the formation of C2α-lactylthiamin diphosphate (LThDP) as an unusually stable ternary complex between the cofactor and pyruvate. The hydroxyaldehyde moiety of GAP was then found to trigger and accelerate decarboxylation of LThDP to give the activated aldehyde. How the second substrate facilitates this reaction remains to be determined but was suggested to involve the repositioning of two arginines acting as a barrier to decarboxylation (Arg478 and Arg420 using E. coli numbering). Alternatively, GAP might reorientate the ternary complex to obtain an optimal dihedral angle for decarboxylation. 2.3. IspC

2.3.1. Discovery. Early work on the discovery and initial steps of the MEP pathway suggested the formation of a 2-Cmethyl-D-erythritol-4-phosphate (MEP) intermediate from DXP by subsequent transposition and reduction reactions. Deuterium-labeled 2-C-methyl-D-erythritol (ME) was indeed incorporated into E. coli isoprenoids such as ubiquinone and menaquinone.35,54,55 Based on these findings, Seto and coworkers initiated a search for the enzyme or catalysts responsible by creating E. coli K-12 mutants auxotrophic for MEP.56 Transformation of these variants with a genome fragment library of the same strain revealed a single open reading frame, the yaeM gene, whose expression was able to rescue the lethal metabolic deficiency. In line with the expected species distribution of the MEP pathway, variants of this gene were detected in the several bacterial genomes that had recently been sequenced. Shortly after, plant homologues were discovered that appeared to be in charge of plastidic isoprenoid production.56,57 Purification and characterization of the corresponding protein product in E. coli revealed that the enzyme is able to catalyze the intramolecular rearrangement of DXP to give 2-C-methylerythrose, which then undergoes 5681


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Figure 7. Rearrangement and reduction of DXP into MEP. IspC catalyzes the isomerization of DXP into 2-C-methyl-D-erythrose-4-phosphate, followed by its NADPH-dependent reduction to MEP. Two mechanisms have been proposed for the first reaction. (a) An α-ketol rearrangement requires deprotonation of the C3 hydroxy group and nucleophilic attack of the C2 on the C4 carbon. (b) The retroaldolization/aldolization reaction involves deprotonation of C4, bond cleavage, and formation of hydroxyacetone enolate and glycoaldehyde phosphate. An aldol reaction between the two intermediates forms the aldehyde common to both mechanisms.

conformational changes of IspC bring cofactor, catalytic metal, and substrate binding sites in close enough proximity for efficient catalysis to occur. 2.3.3. Mechanism. IspC catalyzes the NADPH-dependent isomerization of deoxyxylulose phosphate (DXP) into the aldehyde 2-C-methyl-D-erythrose-4-phosphate, which then undergoes a reduction to give 2-C-methyl-D-erythritol-4phosphate (MEP) and NADP+ (Figure 7). To allow efficient substrate binding and turnover, the enzyme follows a sequential reaction mechanism.63 Prior to catalysis, the NADPH cofactor and metal cation bind to the N-terminal domain and a polar pocket in the active site, respectively. Their binding triggers a conformational change in the C-terminal domain and flexible loop region, during which the latter covers the central domain and creates a protected catalytic pocket. Upon substrate entry, the initial isomerization step occurs, for which two different reaction routes have been proposed.14,16,19,20 The α-ketol (sigmatropic) rearrangement occurs by migration of the C3−C4 bond of DXP to form an aldehyde intermediate (Figure 7a). For this bond-formation reaction, a partial positive charge is required at C2, which can be achieved by protonation or coordination of the keto group by the metal ion. The latter has been suggested to be chelated by the ligand hydroxy groups and act as a Lewis acid.63 Furthermore, a proton is abstracted from the C3 hydroxy group of DXP to form the aldehyde moiety. Deprotonation and bond-cleavage/formation reactions thus result in formation of the branched aldehyde 2-C-methyl-D-erythrose-4-phosphate. An alternative route to this intermediate would be a retroaldolization/ aldolization reaction (Figure 7b). Here, deprotonation of the DXP C4 hydroxy group, followed by initial retroaldol cleavage of the C3−C4 carbon−carbon bond, would result in the formation of a hydroxyacetone enolate and glycoaldehyde phosphate. A subsequent aldol condensation results in the formation of the same aldehyde as obtained by the α-ketol rearrangement. Experiments with deuterium-labeled NADPH and DXP and crystal structures of IspC in complex with the cofactor, substrate, and fosmidomycin inhibitor have provided insights into the subsequent reduction reaction.58,60,64 It was shown that protonation occurs on the intermediate’s Re face when the cofactor transfers a pro-S hydrogen from its nicotinamide C4 to C1 of the aldehyde (Figure 7). IspC is thus a class B dehydrogenase.

and helices, which protrude toward the N- and C-terminal arms of the protein and leave the opposite face of the domain exposed. Upon dimerization, two of these structural motifs associate to form an extended, eight-stranded β-sheet. The central domain further contains a highly flexible loop, which acts as a lid upon substrate and cofactor binding and thus creates a protected active-site cavity. The third and C-terminal domain consists of a four-helix bundle and is characterized by its significant flexibility. It is connected to the central domain by a long linker region, which extends across the complete tip of the protein before entering the first helix of the final motif. Similarly to the central domain, this linker is involved in dimerization. The sequential determination of the IspC apo, binary, tertiary, and quarternary structures allowed an understanding of the significant conformational changes involved in ligand binding (Figure 6b).58,59,61,62 The C-terminal helix bundle and the flexible loop region in particular shift upon binding of NADPH. In the presence of cofactor and substrate (or inhibitor), a 12.5° rotation of the central and C-terminal domains with respect to the N-terminal domain closes the active site to the surrounding solvent. In accordance with this behavior, a decrease in hydrophobic surface areas and a concomitant increase in polar surface areas was observed.58 This induced-fit conformational change is completed with only minor changes in the quarternary structure, where cofactor, inhibitor, and catalytic metal cation are present.61 The IspC active site is formed by the central domain and smaller flanking regions from the N- and C-terminal domains.62 It contains a polar binding pocket in which the metal is fixed in an octahedral coordination sphere by three acidic side chains and three water molecules. In the inhibitor-bound structure, two of the waters are replaced by the oxygen atoms of the fosmidomycin (N-formyl-N-hydroxy) amino group.60,61 Given that the only structure of IspC in complex with deoxyxylulose phosphate available to date contains two diastereomers of the substrate (at C3), its precise binding mode remains unclear. It was shown, however, that the phosphonate, carbonyl, and hydroxy moieties are surrounded by an extensive hydrogenbonding network, which includes residues of the flexible loop region that covers and protects the active site from solvent. Similarly, the carbon backbone interacts with a tryptophan of this same loop.58 Such interactions highlight how the 5682


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Figure 8. Structure and ligand binding of IspD. (a) Analogously to DXS and IspC, the IspD protein forms a homodimer in solution. To achieve this, the subdomain’s protruding arms interlock and form a tight assembly (PDB ID 1INI). (b,c) Each monomer contains its own ligand binding pocket. A glycine-rich loop (highlighted in orange) folds over (b) the nucleoside substrate CTP (PDB ID 1I52) as well as over (c) the CDP-ME product (PDB ID 1INI). The metal ion (Mg2+) is shown as a purple sphere.

were further found to carry plastid-type leader peptides, targeting them to the organisms’ designated sites of biosynthesis. Interestingly, the cytidyl transferase sequence appeared to be translationally coupled to the ygbB gene of as-yetunknown function, and in some cases, the two were fused in a single open reading frame.68 The ygbP protein, later designated as IspD, was purified from a recombinant E. coli host and found to form a homodimer with a molecular weight of 50 kDa in solution.68,69 Its homologue from the plant model system Arabidopsis thaliana was characterized shortly thereafter.70 In activity assays, the enzymes strictly require the presence of divalent metal cations such as Mn2+, Mg2+, or Co2+, with a preference for Mg2+. With respect to its substrate scope, IspD was shown to be highly specific for MEP and to display a clear preference for CTP as the nucleoside triphosphate. In experiments using radioactive phosphorus isotopes, the cofactor’s α-phosphate, but not its βand γ-phosphates, was incorporated. Although the conversion of guanosine triphosphate (GTP) and adenosine triphosphate (ATP) was also observed, it was accompanied by a severe loss in catalytic activity.68,69 The latter two nucleosides are frequently found as substrates and cofactors in enzymatic reactions, whereas the use of CTP is less common. Both the identity and function of this cosubstrate consequently make IspD a highly unusual catalyst and an important target for the selective inhibition of the MEP pathway. 2.4.2. Structure. To date, the three-dimensional structures of IspD from several bacterial species and from A. thaliana have been solved.71−73 They show a strong overall conservation of their tertiary structures and are similar to other nucleosidebinding proteins, in particular, the cytidyltransferases.73 Each monomer of the homodimeric protein shows a characteristic domain structure. The main part of the transferase resembles a Rossmann fold of a seven-stranded β-sheet with interconnected loops and α-helices. Two of the strands are antiparallel and inserted into a five-stranded parallel sheet. A second subdomain is incorporated as a connection between the two antiparallel strands. This so-called β-arm consists of an extended loop that protrudes at a wide angle from the main, globular domain and carries an additional set of two short antiparallel β-strands. With smaller contributions from the main part of the protein, the βarm is responsible for the dimerization of two IspD subunits by forming a hook-like structure that interlocks closely with the neighboring monomer (Figure 8a). The tertiary complex structures of E. coli IspD, Mg2+, and either CTP or CDP-ME have provided valuable insights into ligand binding and catalysis by this enzyme (Figure 8b,c; PDB

The identity of the IspC catalytic mechanism remains controversial. Studies on kinetic isotope effects (KIEs) with deuterium-labeled C3 and C4 hydroxy groups of DXP have supported the retroaldol/aldol mechanism. Secondary KIEs observed with both labels indicate that the corresponding carbon atoms undergo a change in hybridization state from sp3 to sp2 and, in the case of C4, back to sp3, which is not compatible with the α-ketol rearrangement.65,66 These findings, however, are in conflict with the observation that labeled intermediates of the aldol reaction are not converted or incorporated into the substrate. Similarly, neither exogenously added hydroxyacetone nor glycoaldehyde phosphate appear to be recognized by the enzyme.63,67 Although both mechanisms involve the formation of 2-Cmethyl-D-erythrose-4-phosphate as the prereduction intermediate, formation of this aldehyde has never been observed experimentally. It does, however, serve as a good substrate for IspC and can be converted into both DXP and the MEP product. As the equilibrium for isomerization lies on the side of the substrate, it was proposed that this step of the reaction is rate-limiting and directly coupled to the reduction step.63,66 Interestingly, the presence of NADPH appears to be required for both bond migration and reduction, giving the cofactor a double function in this unusual enzymatic reaction. 2.4. IspD

2.4.1. Discovery. The discovery of the MEP pathway’s initial two enzymes, DXS and IspC, allowed the identification and characterization of several downstream catalysts in quick succession. Using [2-14C]-labeled MEP in E. coli cell extracts, Bacher and co-workers were able to observe the formation of a new radioactive product.68 This was subsequently identified as 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) in nuclear magnetic resonance (NMR) spectroscopic assays. A database search for the responsible enzymatic activities, namely, the transfer of cytidine 5′-triphosphate (CTP) onto a second substrate molecule with concomitant loss of its diphosphate moiety, eventually pointed to the unannotated ygbP gene. Using a metabolic engineering approach, Seto and colleagues simultaneously identified the same gene and characterized the CDP-ME product.69 By blocking the biosynthesis between MEP turnover and IPP production in cells complemented with the mevalonate pathway, they showed that ygbP and several other open reading frames play an important role in the reaction sequence. The new protein’s distribution correlated well with the expected presence of the MEP pathway in eubacteria. In plants and Plasmodium species, its homologues 5683


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IDs 1I52 and 1INI). It was shown that both the substrate and product are fixed within the active site by a glycine-rich loop. A large network of hydrogen-bonding interactions was observed between the ligands and side chains, as well as between the ligands and the backbone carbonyl or amide groups of the active site. The cytosine moiety of CTP is recognized specifically, which explains the enzyme’s selectivity and preference for pyrimidine over purine nucleosides. Basic arginine and lysine residues were proposed to position the negatively charged triphosphate moiety (and possibly the 4phosphate of MEP) for catalysis and might further stabilize a potential pentacoordinate transition state during the reaction. A similar function can be assigned to the divalent cation. Although not in direct contact with the enzyme, Mg2+ coordinates all three phosphates of the nucleoside substrate, as well as the α-phosphate of CDP-ME.73 IspD from A. thaliana was the first plant protein structure to be determined for any of the constituents of the MEP pathway and revealed interesting features of the enzyme family’s quarternary structure (PDB ID 1W77).71 Although the homodimers are always related by a 2-fold symmetry axis and residues involved in dimerization are highly conserved, a structural superposition showed significant variations in the subunits’ positions relative to each other. These differences were proposed to influence ligand binding behavior, as well as potential complex formation with IspF, a downstream catalyst of the MEP pathway.71,72 2.4.3. Mechanism. As for the upstream IspC enzyme, two mechanisms were proposed for the IspD-catalyzed transfer of a cytidylyl group from CTP to MEP.73 One option involves the loss of the nucleoside’s β- and γ-phosphates and formation of a metaphosphate intermediate. The latter is subsequently attacked by the 4-phosphate of MEP to produce CDP-ME. Alternatively, the same 4-phosphate might carry out a direct nucleophilic attack on the CTP α-phosphate (Figure 9). The resulting pentacoordinate transition state is unstable and collapses into a diphosphate and CDP-ME. This second, associative mechanism is favored by both mutagenesis and structural data. Based on an analysis of the corresponding complexed structures and an active-site mutant library, Cane and coworkers were able to provide a detailed account of IspD substrate binding and turnover in E. coli.73,74 Using pulse-chase experiments with isotopically labeled substrates, they found that catalysis proceeds by a sequential ordered mechanism (Figure 9a). The reaction is initiated by binding of the CTP nucleoside, followed by MEP as the second substrate. Both molecules and, in particular, their phosphate moieties are recognized, positioned, and stabilized by a network of hydrogen-bonding and ionic interactions. This includes the Mg2+ metal ion, which coordinates all four phosphates involved (Figure 9a,b). Two essential lysine side chains (Lys27 and Lys213) were proposed to position the CTP α-phosphate and MEP 4-phosphate as electron acceptor and donor, respectively, in close enough proximity for the subsequent nucleophilic attack. The latter leads to the formation of a pentacoordinate transition state whose negative charge is, again, stabilized by the metal and positively charged amino-acid side chains. Its collapse leads to the formation of the CDP-ME substrate and the Mg2+-assisted release of the diphosphate leaving group.

Figure 9. IspD mechanism and active-site architecture. (a) Although two reaction types have been proposed, the associate mechanism involving a nucleophilic attack of the MEP phosphate moiety on the αphosphate of CTP, resulting in a pentacoordinated transition state, is better supported by experimental data. The unstable intermediate collapses to give CDP-ME and diphosphate products. (b,c) Ternary complex structures of E. coli IspD with Mg2+ (pink) and (b) CTP (PDB ID 1I52) or (c) CDP-ME (PDB ID 1INI) highlight the ionic and hydrogen-bond stabilization of ligands in the active site. Bond distances of 4 Å or less are shown.

2.5. IspE

2.5.1. Discovery. Knowledge about the distributions of DXS, IspC, and IspD greatly facilitated the discovery of the next downstream catalyst. Even though its function was still unknown, an analysis of whole genome sequences showed that the E. coli ychB gene and its orthologous sequences follow the same pattern of occurrence in eubacteria and plants as other genes of mevalonate-independent isoprenoid biosynthesis. Overexpression of ychB resulted in the production of a 31kDa protein, which was purified to homogeneity and tested for its ability to catalyze a conversion of the cytidylated IspD product. Using 2-14C- or 13C-labeled CDP-ME variants as well as ATP as a cosubstrate in thin-layer chromatography (TLC) and NMR-based assays, Bacher and co-workers observed the formation of a new MEP pathway intermediate: 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-MEP, Figure 11).75 The catalyzed reaction corresponds to a phosphorylation at the substrate’s C2 hydroxy group, and YchB, later designated as IspE, was subsequently shown to resemble 5684


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Figure 10. Fold and active-site architecture of E. coli enzyme IspE (PDB ID 1OJ4). (a) The CDP-ME kinase has a two-domain architecture and α/β fold typical for the GHMP superfamily. Whereas the N-terminal domain (blue) harbors the cofactor binding site, the C-terminal part of the enzyme (green) binds the substrate. At their interface, the two domains jointly form the active site in a deep cleft. (b) An aliphatic pocket and (c) a glycinerich loop ensure selective binding of ATP. (d) The CDP-ME substrate is ideally positioned for proton abstraction by an aspartate side chain acting as the catalytic base and stabilization of the pentacovalent intermediate by a positively charged side chain (here Lys10). (e) Its cytidyl moiety is recognized by purine-specific hydrogen-bonding interactions. For clarity, the cofactor analogue and substrate are labeled only in panel a.

phosphorylating enzymes of the GHMP superfamily in sequence and structure. In addition to galactokinases and homoserine kinases, this family includes mevalonate and phosphomevalonate kinases of the MVA pathway75,76 (hence the designation GHMP). The initial characterization of IspE was confirmed by Kuzuyama et al.’s analysis of E. coli mutants deficient in biosynthetic steps leading from MEP to IPP (as described for IspD in section 2.4.1).77 Their metabolic engineering approach identified the same ychB gene and was followed by the identification and characterization of CDPMEP as a reaction product by high-performance liquid chromatography (HPLC) and NMR analysis.77 2.5.2. Structure. The first three-dimensional structure of an apo IspE protein from Thermus thermophilus was determined in 2003, followed by a ternary complex of the E. coli enzyme with AMP-PNP (a nonhydrolyzable ATP analogue) and the substrate CDP-ME (PDB IDs 1UEK and 1OJ4).76,78 To date, additional X-ray structures from the thermophile Aquifex aeolicus and two mycobacterial species have been deposited in the Protein Data Bank. In line with their overall sequence conservation, the homologues strongly resemble each other and members of their GHMP superfamily. Unlike the latter, which usually assemble into homodimers, most IspE proteins are monomers in solution. Initial crystal structures in which two subunits associate in the asymmetric unit were characterized by minimal surface areas shared between the proteins. Alternative crystal forms later suggested this to be an artifact of crystal contacts rather than dimerization, and size-exclusion chromatography supported the monomeric state in solution, with only minor amounts of dimer formation.76,78−80 Each enzyme is separated into (predominantly) N-terminal and C-terminal domains in charge of cofactor and substrate binding, respectively. Both of these substructures show a characteristic α/β fold with N-terminal helix bundles and extended, twisted β-sheets (Figure 10a).76,78,80,81 The catalytic center is positioned in an open pocket formed at the interface of the two domains. Here, cofactor and substrate are brought into sufficient proximity for transfer of a phosphate group.

Complex structures with nonhydrolyzable AMP-PNP and CDP-ME showed that the substrate’s erythritol end is positioned close to the cofactor’s γ-phosphate. The flexibility of the former in mycobacterial IspE was even proposed to allow for alterations in conformation and insertion depth during the reaction.81 In complex structures, the cytosine moiety of CDPME is sandwiched between two conserved aromatic residues, which provides an explanation for the enzyme’s high selectivity (Figure 10e). Adenosine and uridine derivatives (ADP-ME and UDP-ME, respectively), in which the nucleoside’s cytosine base is replaced, are not accepted as substrates.76 The erythritol C2 hydroxy group forms hydrogen bonds with conserved lysine and aspartate side chains thought to be crucial for its deprotonation and phosphorylation during catalysis (Figure 10d,e).76,78 Binding modes for the phosphorylating cofactor, inferred from complexes with the nonhydrolyzable analogue, appear to be similarly conserved between homologous structures. Both ribose and phosphate moieties are exposed to solvent, with the former being surrounded by a hydrogen-bonding network and the latter being sequestered by a glycine-rich phosphate-binding loop (P-loop). This loop corresponds to one of three highly conserved motifs in the GHMP superfamily, with the other two contributing to the formation of the catalytic pocket. The adenine base is placed in an aliphatic cleft in E. coli and A. aeolicus structures. Here, its less common syn conformation (with respect to the ribose) is stabilized by a hydrogen-bonding network (Figure 10a,c). Despite its requirement for enzymatic activity, no divalent metal cation has been observed in any of the complex structures.76,78,80,81 2.5.3. Mechanism. A catalytic mechanism was proposed for IspE based on the crystal structures of ternary complexes and its similarity to other members of the GHMP family (Figure 11). Both substrate and nucleoside cofactor bind in domainspecific pockets of the enzyme that position them in close proximity for direct transfer of a phosphate group. Although the binding kinetics has not been studied in detail, it has been proposed to follow an ordered-sequential reaction in the closely 5685


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coordinated water molecule in some kinases.76,78,80,84 The exact nature of the metal requirement and function is thus an intriguing question that remains to be answered for members of the IspE protein family. 2.6. IspF

2.6.1. Discovery. When the CDP-ME synthase IspD was discovered as the third enzyme of the MEP pathway, the corresponding ygbP gene was found to be transcriptionally coupled to the unannotated ygbB sequence.68 In some cases, ygbP and ygbB were even fused inside a single open reading frame. Unsurprisingly, the species distributions of the gene orthologues paralleled the presence of MEP-based isoprenoid biosynthesis. To determine its precise function and position within the pathway, Bacher and co-workers cloned and expressed the new ygbB gene in a recombinant E. coli host and purified the corresponding protein.86 When challenged with two upstream products of the MEP pathway, CDP-ME and CDP-MEP, the enzyme was able to catalyze their conversion into the cyclic products 2-C-methyl-D-erythritol2,4-cyclodiphosphate (MEcPP) and 2-C-methyl-D-erythritol3,4-cyclophosphate (MEcP), respectively. Whereas the latter was regarded as an in vitro artifact without physiological relevance, MEcPP had been detected previously as a bacterial metabolite and was now shown to constitute a new intermediate en route from DXP to IPP and DMAPP.86,87 The substrate’s CDP moiety was concomitantly converted into cytidine 5′-monophosphate (CMP). Analogously to IspC, IspD, and IspE, the ygbB gene was also independently identified by a metabolic engineering approach. When its protein product was characterized with respect to CDP-ME turnover by HPLC, mass spectrometry (MS), and NMR analysis, the formation and structures of CMP and MEcPP were confirmed.88 In line with its function and position in the pathway, this new catalyst was subsequently named MEcPP synthase or IspF. The 17-kDa protein associates into a tight homotrimer in solution, with an activity dependent on the presence of two divalent cations, namely, Zn2+ and either Mg2+or Mn2+.86 With the availability of the apo, substrate-bound, and product-bound structures (PDB IDs 1GX1, 1U43, and 1KNJ, respectively), this metal-cofactor requirement was explained, and the enzyme’s mechanism of cyclization and CMP release was determined.89−91 2.6.2. Structure. Shortly after its discovery, three independent structural characterizations were published for the E. coli IspF enzyme and subsequently for its homologues from other bacterial species, namely, the apicomplexa Plasmodium falciparum (PDB ID 4C81) and Plasmodium vivax (PDB ID 3B6N), as well as the plant model system A. thaliana (PDB ID 2PMP).89−93 While differing in space group and composition of the asymmetric unit, all of these crystal structures showed that the enzyme forms a tightly associated homotrimer (Figure 12). Independent of its source organism, this multimeric assembly involves the burial of a large surface area and contributes to the significant stability retained even during denaturing gel electrophoresis and mass spectrometric analysis. Although the enzyme was not assigned to a larger superfamily, trimer formation is the common feature observed between IspF and any of its wider structural or functional homologues.89,90 Within the trimer, each subunit is composed of a fourstranded β-sheet facing three α- and two 310 helices, as well as a

Figure 11. Reaction mechanism of IspE. Phosphorylation of CDP-ME to give CDP-MEP is thought to proceed by way of an associative, inline mechanism. Deprotonation of the substrate leads to the formation of an alkoxide, which, in turn, carries out a nucleophilic attack on the ATP’s terminal phosphate. A pentacovalent intermediate forms and is stabilized by a positively charged Lys side chain before collapsing into the phosphorylated product and ADP.

related mevalonate kinase. Here, the substrate binds first, and its phosphorylated product is equally the first to leave the active site. In homoserine kinases, binding is random, with a minor preference for the cofactor.82,83 Inside the catalytic pocket, phosphorylation is thought to follow an associative in-line mechanism. The C2 hydroxy group of CDP-ME forms hydrogen bonds with the side-chain amino and carboxyl groups of highly conserved lysine and aspartate residues, respectively. Whereas both amino acids polarize the bond, the aspartate acts as a Brønsted base and abstracts a proton. Nucleophilic attack of the formed alkoxide on the ATP cofactor’s γ-phosphate then results in the formation of a pentacoordinate transition state, analogously to the IspD reaction. Interactions with the conserved, positively charged lysine side chain, among others factors, stabilize this intermediate. As the intermediate eventually collapses, CDPMEP and ADP form, and catalysis is complete.76,78,80 For other members of the GHMP superfamily, a divalent metal cation plays an essential role in catalysis. It positions and orients the phosphate moiety in close enough proximity for nucleophilic attack by the acceptor molecule and helps to stabilize the negatively charged, pentavalent transition state.82−85 In addition, activation of the ATP cofactor is facilitated by Mg2+-mediated weakening of the bond between βand γ-phosphate.84 Although it was shown to be required for activity, no metal cation has been observed in any of the IspE crystal structures determined to date. A highly conserved glutamate positioning the metal in GHMP proteins is equally not conserved. Its role, however, might also be fulfilled by a 5686


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Figure 12. Tertiary and quarternary structures of E. coli IspF (PDB ID 1H48). (a) Each monomer is composed of one four-stranded β-sheet and one flexible two-stranded β-sheet, facing a set of α- and 310 helices. (b) In solution, IspF assembles into a tight homotrimer, which is also reflected in its crystal packing. The three active sites, shown here with the two products MEcPP and CMP, are each shared between two subunits. (c) A surface view highlights the tight assembly between monomers, as well as the access points to the catalytic sites. (d) For some homologues including the E. coli enzyme, downstream isoprenoids such as GPP were shown to bind at the bottom of a cavity. Here, they are coordinated by three arginine residues, one from each subunit. The inlet shows a side-on representation of the bound terpenoid.

Figure 13. Reaction mechanism and active-site architecture of IspF. (a) The enzyme catalyzes an intramolecular cyclization reaction of CDP-MEP in which the MEP moiety’s 2-phosphate carries out a nucleophilic attack on the CDP β-phosphate. A pentacovalent transition state forms whose collapse leads to the formation of the products MEcPP and CMP. Zn2+ (gray) and Mg2+ (pink) or Mn2+ ions stabilize the negative charges of the substrates, reaction intermediates, and product. (b) In the E. coli IspF active site, the metal ions are found in tetrahedral (Zn2+) and octahedral (here, Mn2+) coordination spheres formed by highly conserved active-site residues and water molecules. Complex structures of the enzyme have been obtained with both products (right) and the CDP component of the substrate (left). Both highlight how the defined ligand and metal cofactor binding enables catalysis (PDB IDs 1GX1, 1H48).

highly flexible section of two short antiparallel β-sheets (Figure 12a). The latter is the so-called β-flap that covers the active site upon substrate binding. In its multimeric state, the enzyme’s βsheets, although slightly tilted with respect to each other, face their respective counterparts and thus create an internal, predominantly hydrophobic channel. In early structures, one end of this cavity appeared to be closed off by a fragment of unidentified electron density, which was, in turn, coordinated

by an arginine side chain from each of the three subunits. This density was initially assigned as a sulfate or phosphate ion or, alternatively, proposed to constitute a downstream isoprenoid product carrying a diphosphate moiety (Figure 12b−d).89−91,94 The latter would imply a possible feedback regulation of the enzyme.94 With the high-resolution structures of Shewanella oneidensis IspF (PDB ID 1T0A) and E. coli IspF (PDB ID 1H48), coordination of farnesyl pyrophosphate (FPP) and 5687


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Figure 14. Structure and reactions of a bifunctional IspDF enzyme (PDB ID 1W57). (a) The E. coli IspDF protein is separated into an N-terminal domain (green) and a C-terminal domain (blue), corresponding to the IspD and IspF components, respectively. Their two active sites catalyze the cytidylation of MEP to CDP-ME and the intramolecular cyclization of CDP-MEP to MEcPP, respectively. (b) In solution, the enzyme forms a large hexameric assembly, which is shown with the individual monomers labeled. This quarternary structure does not, however, apply to all bacterial homologues.

to close over the substrate and protect it from the surrounding solvent. Inside the catalytic cavity, both the CDP and MEP substructures are recognized specifically (Figure 13, bottom). In addition to hydrogen-bonding and hydrophobic interactions with amino-acid side-chain and backbone atoms, this requires the active site’s two metal cations to align the diphosphocytidyl moiety. The interaction with Zn2+ in particular increases the electrophilic character of the β-phosphate in preparation for a subsequent in-line nucleophilic attack by the MEP moiety’s 2phosphate. To enable the formation of an eight-membered ring, the otherwise flexible substrate is bound in highly defined positions that bring the electron donor and acceptor in close proximity.90 The negative charge of a cyclic transition state is compensated by metal-ion coordination until it collapses, at which point the CMP moiety is released from the cyclic product and catalysis is complete (Figure 13).89−91 2.6.4. Bifunctional IspDF Enzymes. When the first open reading frames encoding IspD and IspF enzymes were discovered, it was noted that several bacterial species have the two genes fused into a single sequence.68 This fusion was predominantly observed for α-, ε-, as well as δ-proteobacteria, whereas β- as well as γ-proteobacterial genomes encode the individual enzymes.97,98 The recombinant expression and mechanistic and structural characterization of the joint gene products revealed an unusual set of bifunctional IspDF proteins. The first analysis was carried out on the Campylobacter jejuni enzyme by Hunter and coworkers.97 The purified enzyme was shown to convert MEP into CDP-ME and CDP-MEP into MEcPP, both in a divalent metal-dependent manner. The phosphorylation reaction typically catalyzed by IspE was not observed, however. Instead, bifunctional IspDF enzymes from C. jejuni and Agrobacterium tumefaciens were found to form a complex with the kinase, which allows efficient synthesis of MEcPP from CDP-ME. Interestingly, such a multienzyme assembly was also observed for the monofunctional E. coli enzymes.99 The spatial proximity was proposed to improve the biosynthetic efficiency by providing a shorter route for substrates and products between the catalysts, as well as a potential control over metabolic flux.

geranyl pyrophosphate (GPP) molecules, respectively, was verified. The A. thaliana cavity structure or the complete absence of a channel in the Thermus thermophilus enzyme would, on the other hand, preclude any such binding.92,94−96 Consistent with the requirement for multimeric assembly, the IspF active site is situated at the interface of two subunits, with side chains from both contributing to the catalytic center (Figure 12 d, Figure 13). Here, the substrate and reaction intermediate are positioned and stabilized in conformations that are optimal for catalysis by interactions with amino acids and with two distinct essential metals. A zinc ion, itself tetrahedrally coordinated by an aspartate and two histidine residues as well as the β-phosphate of MEcPP, positions the cytidyl moiety of the substrate. In the absence of the metal, a corresponding shift of 1.7 Å is observed for this part of the substrate, and mutations of the coordinating residues render the protein inactive. A second cation, either Mg2+ or Mn2+, was found to arrange and stabilize both the α- and β-phosphates of the CDP substructure. It is coordinated in an octahedral sphere by the aforementioned phosphates, three water molecules, and a glutamate side chain. The substrate’s MEP moiety is held in a stable position by hydrophobic interactions with an isoleucine side chain, whereas the terminal 2-phosphate interacts with the backbone and side chain of a histidine and a serine in the active site.89−91 Therefore, MEcPP, which would be significantly more flexible in solution, is in an ideal conformation for an intramolecular nucleophilic attack of the MEP 2-phosphate on the CDP moiety’s β-phosphate, as described below.91 2.6.3. Mechanism. IspF catalyzes the intramolecular cyclization of CDP-ME to MEcPP, with the concomitant loss of a CMP moiety, by means of an associative in-line mechanism (Figure 13). Analogously to IspD and IspE, the reaction involves the nucleophilic attack on a phosphate moiety, formation of a pentacoordinated transition state, and collapse of the latter to form the two products. Detailed information on ligand recognition, positioning, and activation was gained with the elucidation of CDP nucleoside- and product-bound complex structures, particularly for the E. coli enzyme.89−91 As CDP-MEP binds to the active-site cleft formed by two IspF monomers, a flexible loop, the so-called β-flap, is thought 5688


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Figure 15. IspG quarternary structure and conformational changes. (a) In the bacterial IspG enzymes, shown here from the thermophile T. thermophilus, two monomers associate in a head-to-tail arrangement to form the active homodimer. The subunit separation is indicated by a dashed line (PDB ID 4G9P). (b,c) When IspG structures were determined in their holo and ligand-bound forms (light green and dark green, respectively), a significant conformational change was observed. A 60° rotation of the C-terminal domain closes the active site to surrounding solvent and brings the substrate and inorganic cofactor in close enough proximity for catalysis (PDB IDs 4G9P, 2Y0F).

such as yeast. As before, disruption of the gene was lethal, and only E. coli test systems supplemented with the mevalonate pathway allowed for the identification of otherwise lethal mutants.102,103 In isotope-labeling experiments with 13C-deoxyxylulose and recombinantly overexpressed genes of all known and putative MEP pathway constituents, the gcpE-dependent production of a new reaction intermediate was observed. NMR analysis identified the molecule as 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP), which would be obtained by reductive deoxygenation of the IspF cyclization product MEcPP. The GcpE protein was consequently renamed IspG, being the next, and presumably penultimate, catalyst in IPP and DMAPP biosynthesis.104 Although initial recombinant expression and purification of the enzyme proved straightforward, the observed activity remained comparatively low. The presence of three highly conserved cysteines and similarities with sequence motifs from ferredoxin and aconitase enzymes hinted at the presence of a catalytic [4Fe4S] cluster.104,105 When protein purification and activity assays were subsequently carried out under anaerobic conditions and in the presence of a reducing agent, the efficient turnover of MEcPP to HMBPP was indeed observed. Furthermore, IspG showed a shoulder at 413 nm in its ultraviolet−visible (UV−vis) absorption spectrum, another feature characteristic of [4Fe4S] cluster proteins. Similar observations were made after anaerobic reconstitution, which restored activity to the enzyme.106,107 In the row of unusual reactions catalyzed by members of the MEP pathway, IspG thus constituted the first example of an iron− sulfur enzyme. More remarkable still was its unprecedented ability to carry out the reductive elimination of a hydroxy group.104 Compared to many of the constituents of the upstream pathway, this oxygen sensitivity and complex reaction of IspG rendered determination of its structure and mechanism challenging yet highly fascinating. 2.7.2. Structure. The first IspG structure was determined in 2010 and showed the holo protein of the thermophile Aquifex aeolicus in its [4Fe4S] cluster-bound form (PDB ID 3NOY).108 Within the crystal, the four molecules in the asymmetric unit

However, no evidence for active substrate channeling was found.99,100 When the first, and to date only, IspDF crystal structure of the C. jejuni enzyme was determined, it showed a clear twodomain architecture in which the N- and C-terminal parts of the protein correspond to IspD and IspF, respectively (Figure 14a).99 Analogously to the monofunctional enzymes, the latter forms a catalytic cavity through the close assembly of its βsheets. In the unit cell, the IspD component is positioned near a crystallographic 2-fold axis, whereas the IspF domain is close to a 3-fold axis (Figure 14b). Although the resulting hexameric assembly of three dimers and two trimers was observed both in the crystal and in solution, it does not seem to apply to all IspDF homologues. The corresponding enzyme from the Gram-negative bacterium Mesorhizobium loti, for example, was mostly observed in its dimeric form.98,99 Although many examples of bifunctional enzymes exist in nature, catalysis of two nonconsecutive pathway reactions within a single protein as for IspDF is rare.98 This feature makes them valuable, selective drug targets with a fascinating, unexplored enzymology. 2.7. IspG

2.7.1. Discovery. Whereas many upstream catalysts were discovered and characterized in a sequential manner and short succession, the last two reaction steps of the MEP biosynthetic pathway proved to be more challenging. As early as 1992, an unannotated open reading frame was discovered in the E. coli genome.101 It was shown to be essential to cell survival, but aside from its apparent transcriptional association with a histidyl tRNA synthetase, nothing was known about its function. It was named “gene coding for protein E”, or gcpE for short, and was predicted to encode a protein with a molecular weight of approximately 40 kDa. An association between the gcpE gene and other methylerythritol phosphate pathway enzymes was discovered in 2001 when, through a bioinformatics approach, they were found to share their characteristic distribution patterns. As with DXS and IspC− IspF, the unannotated sequence was detected in various bacterial species, plants, and apicomplexa, but not in eukaryotes 5689


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Figure 16. Crystal structures of ligand-bound T. thermophilus IspG. Co-crystal structures were determined with (a) its substrate (PDB ID 4G9P), (b) the carbocation/radical intermediate (PDB ID 4S3C), (c) the carbanion intermediate (PDB ID 4S3B), and (d) the HMBPP product (PDB ID 4S23). Each ligand is bound in a clearly defined pocket. The diphosphate moiety is surrounded and stabilized by the positively charged side chains of arginines and a lysine (shown in panel a). Two glutamate and an asparagine residue were proposed to be involved in a proton relay chain during the final stages of catalysis.

Figure 17. Two-electron reduction of MEcPP to HMBPP by IspG. As the circular substrate binds, the enzyme adopts a closed conformation in which the diphosphate moiety is recognized and stabilized by a positively charged pocket. Prior to catalysis, highly transient, nonreductive ring opening takes place. The subsequent entry of two electrons, shuttled by the iron−sulfur cluster, leads to the formation of carbocation/radical and carbanion intermediates. The former may exist in equilibrium with a ferraoxetane transition state. Elimination of the oxygen from the final intermediate occurs upon protonation, after which the release of water and the linear product completes catalyst turnover. An epoxide derivative of HMBPP, which is not thought to constitute an actual reaction intermediate, is converted into MEcPP and HMBPP under oxidizing and reducing conditions, respectively. The amino-acid numbering corresponds to the numbering of the T. thermophilus enzyme (see Figure 16).

N-terminal pole by two small, antiparallel β-strands. In contrast, the opposite end of the cavity is open and solvent-exposed. The C-terminal domain is significantly smaller and consists of a fivestranded β-sheet sandwiched between three α-helices. Subsequently, a variety of structures were published for the Thermus thermophilus IspG homologue, either in its holo form

assemble into two sets of characteristic homodimers in which each monomer is aligned with its inverted counterpart on a longitudinal axis (Figure 15a). Each monomer can, in turn, be subdivided into two domains. The N-terminal domain takes the shape of an eight-stranded TIM barrel that is surrounded by a uniformly spaced set of α-helices. The barrel is closed off at its 5690


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group, allowing formation of an Fe−O bond. Although the distance between the substrate and the amino acid’s carboxylate moiety is unusually large, the basicity of the latter is increased by the presence of a proton relay chain in the active site. This involves the first (displaced) glutamate, as well as an asparagine side chain (Figure 16).108,110,113 Once bound, the bond between the MEcPP substrate’s C2 carbon and the terminal phosphate transiently breaks and reforms, as shown by isotopic-exchange experiments with the diphosphate moiety’s oxygen atoms. The resulting ring opening occurs in the absence of any reducing agents.120 This leads to the formation of a cation species on the C2 carbon, which can alternate with a radical form through an internal electron transfer (IET). This transfer is likely to be accompanied by a change in the iron−sulfur cluster’s oxidation state from 2+ (cation) to 3+ (radical) (Figure 17c1,c2).111,115,120 Early investigations into IspG catalysis suggested the formation of an epoxide transition state prior to any cluster-mediated reduction (Figure 17c3).116 Such an intermediate was indeed shown to be accepted as a substrate by the enzyme. It is converted back into MEcPP under oxidizing conditions, reacts to form HMBPP product under reducing conditions, and shares identical EPR spectroscopic intermediates with the educt.117,118,121,122 As all efforts to show epoxide formation from MEcPP were unsuccessful, however, the former is now considered an artificial substrate rather than a natural reaction intermediate. In cocrystal structures, the surrogate serves as a suitable ligand for trapping the highly transient carbocation/ radical state (Figure 17c). IspG catalysis is fully initiated with the first single-electron transfer. When the electron is obtained from an external donor and shuttled into the substrate by the [4Fe4S] cluster, the MEcPP ring opens permanently. Either radical or carbocation species form on the C2 carbon with the concomitant change in cluster charge described for the transient, prereduction state. Both forms can be interconverted by IET (Figure 17d1,d2). The carbocation state has been proposed to give rise to a ferraoxetane species in which a bond is formed between the cluster’s apical iron and the substrate’s C2 carbon and C3 oxygen (Figure 17d3). Such an organometallic intermediate has not been observed in any of the crystal structures determined to date, and its existence remains controversial. Although it is supported by extensive EPR spectroscopic analyses and can exist in equilibrium with the singly reduced cation species, objections have been raised against the iron−carbon bond location and kinetic competency of this intermediate.111,117,118,123 A second electron transfer to the C2 carbon radical leads to the formation of a carbanion species without a change in the oxidation state of the [4Fe4S]2+ cluster (Figure 17e). In the last reaction step, formation and release of the HMBPP product requires breakage of the iron−oxygen bond between metal cofactor and ligand (Figure 17f). This deoxygenation reaction is facilitated two-fold by the development of a partial positive charge on the carbanion intermediate’s C3 carbon and coordination of the oxygen by the carboxylic acid moiety of a glutamate side chain. An E1cB elimination reaction then results in the formation of the HMBPP product, and protonation of the Fe−OH group by the relay chain described above provides an efficient H2O leaving group. Product release completes catalysis and returns the protein to its flexible, open-closed state.111 Several native reducing systems have been proposed to supply the electrons required for this complex and unusual

with the bound [4Fe4S] cluster (PDB ID 2Y0F) or in tertiary complexes with the cluster and substrate, reaction intermediates, product, or inhibitor molecules.109−111 All structures resemble their A. aeolicus counterpart closely in terms of fold and multimeric assembly and share their N-terminal domain architecture with a third homologue from Bacillus anthracis (PDB ID 4MWA). For the latter, however, the C-terminal component of the protein is absent from the crystal structure, and no further data on the enzyme have been published to date. Certain IspG homologues, such as those of plants and P. falciparum, have an additional domain between the N- and Cterminal parts of the protein. This was proposed to fold into a second TIM barrel and allow the enzymes to function as monomers in solution.112,113 The IspG active site is composed of two parts, which are contributed from the domains of neighboring monomers.108 Two flexible loops between the C-domain’s β-sheet and its adjacent helices surround the [4Fe4S] cluster and position it between the N- and C-terminal domains. Here, the inorganic cofactor is covalently bound to four highly conserved aminoacid residues, namely, three cysteines and a glutamate. Whereas the cysteines are part of widely conserved iron−sulfur cluster binding motifs, the glutamate was the first of its kind to be observed in an enzyme:[4Fe4S] complex. The second part of the active site is situated at the N-domain’s C-terminal pole. Its composition of largely positively charged amino acids ensures efficient binding of the MEcPP’s phosphate moiety (Figure 16).110 As the metal cluster and substrate binding site are spatially too far apart for catalysis in the holoenzyme, a significant structural rearrangement of the dimer must occur upon ligand binding.110 This process of “induced fit” involves a rotation of the C-terminal part of IspG around the flexible loop connecting the two domains (Figure 15b,c). It thus enables direct contact between the cluster and the MEcPP binding site, which are contributed from separate monomers.110 With this significant conformational flexibility, dimeric IspG is thought to exist in a constant equilibrium between its open (holo) and closed (ligand-bound) conformations.111,114 This property ensures the efficient catalysis described below. 2.7.3. Mechanism. Despite the identification of its substrate and product and the elucidation of its threedimensional structure, the reaction mechanism of IspG remained controversial. Although it was known to involve a [4Fe4S] cluster-mediated, two-electron reduction process and elimination of the MEcPP C3 hydroxy group, identification of the reaction intermediates proved challenging. Various combinations of carbocation, radical, carbanion, and ferraoxetane species were proposed to be involved as potential transition states. A combination of findings, particularly from isotopic-exchange experiments, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography, eventually permitted a detailed description of the IspG reaction mechanism (Figure 17).106,107,115−119 As the MEcPP substrate binds, the enzyme adopts its closed conformation by means of a hinge movement around the flexible loop connecting the two domains. For the T. thermophilus enzyme, this was shown to involve a rotation of more than 60° (Figure 15). This structural change displaces the glutamate side chain coordinating the [4Fe4S] cluster’s fourth iron. At the same time, the substrate is positioned in close enough proximity for the formation of a covalent bond with the apical iron (Figure 17b). To enable the latter, a second glutamate is thought to deprotonate the MEcPP C3 hydroxy 5691


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Figure 18. Tertiary structure, cofactor binding, and ligand binding of IspH. (a) The enzyme is separated into three highly similar domains, which assemble into a propeller-like structure around the iron−sulfur cluster (PDB ID 3KE8). (b) Upon substrate binding, the rotation of one domain is required for catalysis. The A. aeolicus open conformation (green) was superimposed on a closed E. coli diphosphate complex (blue). A conserved SXN motif above the iron−sulfur cluster is highlighted in beige (PDB IDs 3DNF, 3KEL). (c−f) X-ray structures of E. coli enzyme−ligand complexes display (c) cysteine coordination of the [4Fe4S] cluster and binding of the HMBPP diphosphate moiety in a polar pocket (PDB ID 3KE8), (d) the alkoxide intermediate (PDB ID 3KE8), (e) rotation of the methyl hydroxy group and hydrogen-bond formation with the catalytic acid/base trapped in a Glu126Gln mutant (PDB ID 3SZU), (f) binding of IPP (PDB ID 3KEM), and (g) binding of DMAPP (PDB ID 3KEF). The final two images include a structurally conserved water formed during the reaction.

subsequently confirmed.116,126,129 In feeding experiments with [U−13C5]-labeled deoxyxylulose, the enzyme was found to convert the linear IspG product HMBPP into IPP and DMAPP in a ratio of 6:1. This reaction, corresponding to a two-electron reduction and OH-group elimination analogous to that of IspG (Figure 19), thus constitutes the final step of the MEP pathway, and LytB was consequently renamed IspH. Monomeric and dimeric IspH homologues from a variety of organisms have since been characterized, including those of several bacterial and apicomplexan pathogens, as well as the plant model system A. thaliana. As for IspG, the presence of a set of highly conserved cysteines and a loss of activity in purified enzymes hinted at the involvement of an iron−sulfur cluster and additional redox partners in catalysis.116,130 In accordance with this suggestion, the use of anaerobic protocols allowed efficient purification, reconstitution, and activity assays for IspH.131 UV−vis and EPR spectroscopic analyses confirmed the stoichiometry of its prosthetic group as a [4Fe4S] cluster typical for enzymes catalyzing single-electron reduction reactions.132 In combination with the structural data available for IspH from E. coli, the thermophile Aquifex aeolicus, and Plasmodium falciparum, these findings have provided detailed insights into the second, equally fascinating iron−sulfur catalyst of the MEP pathway. 2.8.2. Structure. After the identification of an iron−sulfur cluster as their prosthetic group, anaerobic purification and crystallization protocols enabled the structure determination for two bacterial IspH homologues from A. aeolicus and E. coli, as well as the apicomplexan P. falciparum (PDB IDs 3DNF, 3F7T,

reaction of the isoprenoid biosynthesis pathway. These include flavodoxin/flavodoxin reductase in E. coli, as well as ferredoxin/ ferredoxin reductase systems in plants.107,116,124 2.8. IspH

2.8.1. Discovery. As for the upstream IspG, the unusual reaction and cofactor requirement of the MEP pathway’s final catalyst proved challenging for its purification and kinetic and structural characterization. When the enzyme’s corresponding gene sequence, designated lytB, was characterized in 1997, it was known only to be involved in conferring resistance to the antibiotic penicillin.125 Homologous open reading frames were initially discovered in E. coli, Haemophilus inf luenza, and Synechocystis species and later found to be consistent with the presence of other methylerythritol phosphate pathway members. Plastid targeting sequences for plants and apicomplexa even hinted at the expected subcellular localization in these organisms.126 A connection between the LytB protein and the final stages of the MEP pathway was made by Cunningham et al. in 2000.127 Insertions into the gene sequence proved lethal to the cyanobacterium, and its protein product appeared to influence the ratio of IPP to DMAPP in isoprenoid biosynthesis. E. coli cells deficient in the active enzyme would form spheroblasts because of the lack of isoprenoids required in the production of bactoprenols.128 The function of these lipids in cell-wall biosynthesis also explained the initial observation of lytB involvement in penicillin resistance. A proposal that the LytB protein is involved in a reaction following the formation of the cyclic IspF product MEcPP was 5692


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Figure 19. IspH-mediated formation of IPP and DMAPP from HMBPP. (a) Substrate entry induces a conformational shift in one of the enzyme domains. (b) As HMBPP binds to the active site in a bent conformation, its diphosphate moiety is coordinated by hydrogen bonding to a polar pocket (outlined in blue). An alkoxide complex (intermediate I) forms between the substrate’s C1 hydroxy and the [4Fe4S] cluster’s apical iron. (c) Upon the first single-electron reduction of the cluster, the hydroxy group rotates away and forms a hydrogen bond with the strictly conserved glutamic acid, and the metal cofactor and ligand are connected in a weak π complex (intermediate II). (d) After protonation by glutamic acid, elimination of water and a formal two-electron reduction of the substrate lead to the formation of the allyl anion intermediate III bound to an oxidized HiPIP [4Fe4S]3+-like cluster. (e) A second single-electron reduction and C3 or C1 carbons by the diphosphate moiety eventually give rise to IPP and DMAPP, respectively.

form, cluster integrity is ensured by its coordination to three water molecules.132,138 As mentioned above, the IspH active site is positioned in a hydrophobic pocket at the interface of the individual domains (Figure 18). Here, three highly conserved cysteine residues coordinate the iron−sulfur cluster. Comparisons between the X-ray structures of an E. coli enzyme−diphosphate complex and A. aeolicus holoenzyme show that ligand binding causes a 20° tilt of one of the domains.133 This conformational change was proposed to contribute to the opening and closing of the active site for catalysis. In tertiary complexes with substrate, the HMBPP diphosphate moiety is coordinated in a polar pocket and surrounded by an extensive hydrogen-bonding network (Figure 18c).139 It is composed, among others, of conserved histidine, serine, glutamate, threonine, and arginine side chains. The entire HMBPP molecule is bound in a bent, hairpin conformation, with its allylic carbon chain sandwiched between the diphosphate group and the iron−sulfur cluster. Its C1 oxygen forms a covalent bond with the [4Fe4S] cluster’s apical iron. Through direct coordination to a threonine side chain, the resulting alkoxide is embedded in the elaborate hydrogenbonding network described above. Here, the close proximity to a glutamic acid proton donor and relay system ensure its efficient hydrogenation and, ultimately, elimination from the C1 carbon (see section 2.8.3). It is interesting to note that the closed IspH form was found to be particularly stable when

and 4N7B). Whereas the latter two exist as monomers in solution, the former is predicted to assemble into a homodimer by the macromolecular interface prediction software PISA.133−136 All three proteins resemble each other closely in their overall, unprecedented fold and propeller-like arrangement of three α/β domains. Each domain is composed of a central β-sheet sandwiched between two sets of α-helices (Figure 18a). At the interface, a hydrophobic pocket is formed, which harbors the inorganic cofactor. In the thermophile A. aeolicus, this pocket is protected from surrounding solvent through dimerization, whereas the E. coli and P. falciparum counterparts are covered by an additional extended C-terminal loop containing a short β-sheet positioned above the prosthetic group. This belt-like structure is unique to certain classes of IspH homologues and was proposed to influence interactions with redox partners, oxygen sensitivity, and electron-transfer rates.137 The iron−sulfur cluster itself varies between [3Fe4S] and [4Fe4S] stoichiometries in the crystal structures. Whereas the holoenzyme, product-bound, and diphosphate-bound complexes contain the open, three-iron form of the cluster, the HMBPP substrate binds to the closed, four-iron, cage-like cofactor. Subsequently determined structures of IspH binding to inhibitor molecules display both variants. Spectroscopic analyses have since shown that native IspH employs the full [4Fe4S] for catalysis, where, even in the enzyme’s substrate-free 5693


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reduction and protonation subsequently led to product formation and release from the [4Fe4S]2+ prosthetic group (Figures 18f,g and 19e). The hydrogen required for this final step might be provided by the intermediate’s own diphosphate moiety. Because of steric constraints imposed by the iron− sulfur cluster, protonation is restricted entirely to the Si-face of intermediate III. This occurs preferentially on C3, but also on C1, and consequently yields the two products IPP and DMAPP at a ratio of 6:1.132,139 Their release from the enzyme might be facilitated by the buildup of negative charges inside the polar pocket, especially on the catalytic acid and the products’ diphosphate moieties.139,141 Whereas in vitro assays frequently make use of chemical reducing agents such as dithionite and methyl viologen, IspH homologues were shown to accept electron-transport systems such as flavodoxin or ferredoxin and their respective reductases as natural redox partners.132,144 In a study using diquarternary salts as artificial reducing systems, IspH activities could be improved by more than 100-fold, and the iron−sulfur cluster’s redox potential was proposed to act as a natural regulator of IspH activity. This property would also explain the large variation in IspH activities observed experimentally.145 It is noteworthy that, in general, experimental data on the oxidation states of the iron−sulfur clusters as well as on the presteady-state enzyme kinetics are still scarce for both IspG and IspH. The mechanisms outlined here thus represent the most recent models. These were developed from a variety of earlier proposals and might still be adjusted in the future as more data become available, particularly from structural and spectroscopic studies. In addition to their role as the final reductases of the MEP pathway, IspH homologues also display promiscuity in their substrate tolerance and activities. They were shown to hydrate acetylenes into aldehydes and ketones and also to be able to catalyze the reductive conversions of HMBPP to isoprene and of DMAPP to isoamylene.144,146 These findings add to the list of unusual structural and mechanistic features of this last catalyst in the biosynthesis of isoprenoid building blocks. They also expand the opportunities for the design of selective drugs targeting this pathway, as summarized briefly in the following section.

HMBPP is bound, shielding its substrate from the effects of oxidizing environments.139 Although the covalent Fe−O bond is broken and the alkoxide moiety is absent in complex structures between IspH and its reaction intermediates and products, the overall ligand conformation and active-site coordination remain unchanged. In combination with computational modeling and spectroscopic methods, these structural features eventually provided crucial information for the elucidation of the controversial IspH reaction mechanism. 2.8.3. Mechanism. As for the preceding catalyst IspG, elucidation of the IspH reaction mechanism was complicated by the enzyme’s oxygen sensitivity and associated challenges in purification and characterization. The conversion of HMBPP to IPP and DMAPP was known to involve a two-electron reduction reaction and concomitant elimination of the substrate’s hydroxy group. The identity of reaction intermediates, however, long remained controversial. Radical, cationic, anionic, and ferraoxetane transition states were proposed for the allylic carbon moiety but could not be identified unambiguously.116,118,129,131,132 Although initial proposals suggested IspH to follow a Birch reduction-like mechanism, more recent findings from isotopic-labeling, spectroscopic, and crystallographic studies favor the bioorganometallic reaction described below (Figures 18 and 19).118,119,140−142 In its holo state, prior to ligand binding, IspH contains a [4Fe4S]2+ cluster as its prosthetic group (Figure 19a).143 Upon entry of HMBPP into the active site, hydrogen-bonding interactions between polar amino-acid side chains and the substrate’s diphosphate group lead to a conformational change that closes the catalytic pocket and protects it from surrounding solvent (Figure 19b). This reaction is crucial as it favors the enzyme-catalyzed reductive dehydroxylation over the solventmediated hydrolysis of the diphosphate.141 The HMBPP C1 hydroxy moiety then binds to the iron−sulfur cluster’s apical iron to form an alkoxide complex (intermediate I, Figures 18d and 19b). As the cofactor is reduced from a [4Fe4S]2+ to a [4Fe4S]+ state by an external electron donor, a reduction in the apical iron’s Lewis acidity was proposed to favor breakage of the iron−oxygen bond. The substrate’s free hydroxymethyl group subsequently rotates away from the cluster and forms hydrogen bonds with a catalytic glutamic acid and its own diphosphate moiety (Figures 18e and 19c). Rather than the previously proposed covalent Fe−O or ferraoxetane-type Fe−C bonds, the iron−sulfur cluster is now thought to form a weak π or van der Waals complex with the substrate (intermediate II, Figure 19c).141,142 This second intermediate can be protonated at the C1 OH position by the glutamic acid, which enables the elimination of water. A formal two-electron reduction of the substrate leads to the formation of the allyl anion intermediate III bound to a high-potential iron−sulfur protein (HiPIP) type of [4Fe4S]3+ cluster (Figure 19d). Alternatively, this might already correspond to a protonated form of the allyl anion and constitute a [4Fe4S]3+-like product complex.118 The unusual oxidation state of the cofactor was previously proposed for IspG by Duin and co-workers, who also highlighted analogies with the mechanism of ferredoxin:thioredoxin reductase.123 For IspH, Oldfield and co-workers obtained EPR spectra favoring the HiPIP type over the [4Fe4S]+ cluster.142 Using IspH mutant and wild-type proteins, they were also able to trap paramagnetic intermediates II and III and showed that their identities were inconsistent with the radical formation required for a Birch reduction-like mechanism. A second single-electron

3. DRUG TARGETS IN THE METHYLERYTHRITOL PHOSPHATE PATHWAY OF ISOPRENOID BIOSYNTHESIS One of the key features of the methylerythritol phosphate pathway is its characteristic species distribution. Whereas MEP catalysts are found in a number of Gram-positive and most Gram-negative bacteria (including cyanobacteria) and several apicomplexan species, they are completely absent in mammals. Plants make use of both pathways in a compartmentalized manner: The mevalonate pathway enzymes are predominant in the cytosol, whereas the MEP pathway is the key route to many isoprenoids synthesized in chloroplasts. This species selectivity and prevalence in many notorious pathogens renders enzymes of the MEP pathway highly attractive drug targets, which have been the subject of numerous in silico, in vitro, and in vivo inhibition studies. Detailed descriptions of their inhibition would exceed the scope and focus of this review and have been published in detail elsewhere.118,147,148 Nonetheless, a short overview on the general druggability of the MEP pathway and a selection of its more prominent examples is outlined below. Representative inhibitors and their targets are listed in Tables 1 and 2. 5694


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Table 1. Enzymes of the MEP Pathway and Their Catalyzed Reactions along with a Selection of the Best-Studied Inhibitors/ Inhibitor Classes and Their Respective Binding Pockets in the Enzymea

a

Binding pockets marked with a superscript a have been inferred from experimental data or computational modelling but have not been verified crystallographically.

Table 2. Core Structures of Representative Inhibitors Targeting Enzymes of the MEP Biosynthetic Pathwaya

a

R = possible site of modifications, X = carbon or nitrogen atom.

Inhibition of MEP isoprenoid biosynthesis faces a number of general problems, which have been addressed to various degrees for the individual enzymes.147 Many of the catalytic pockets are highly polar, as they need to accommodate charged mono- or diphosphate moieties of substrates and/or cofactors. This causes problems in ligand design, for which a higher degree of lipophilicity is typically required. Replacing the

inhibitors’ diphosphate moieties with other functional groups can improve their drug-like properties, such as bioavailability, but often leads to a concomitant decrease in their potency.149 In addition, inhibitors mimicking or targeting the binding of cofactors such as ATP, CTP, and iron−sulfur clusters are prone to binding other enzymes and can cause cytotoxicity in the host organism. In such cases, the identification of binding features 5695


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homologues, drawbacks such as the cytotoxicity of pseudilins still need to be addressed. For the next downstream catalyst, IspE, a set of first- and second-generation inhibitors was developed. In particular, the cytidine derivatives assayed by Diederich and co-workers display activities down to a nanomolar range and were shown to bind the enzyme’s substrate binding pocket and not that of the nucleotide.161,162 Such binding properties are essential in view of potential selectivity issues with related kinases, particularly with other members of the GHMP family.147 The cytidine analogues and a set of more recently characterized pyrazolopyrimidine derivatives inhibit both IspD and IspE homologues, which might prove highly valuable in preventing the development of resistance in targeted pathogens.163 The MEP pathway’s fifth enzyme, IspF, was predicted to be one of the most druggable catalysts because of the comparatively high ratio of apolar amino acids and resulting lipophilic character of its active site.164 A potential regulatory role in the biosynthetic pathway renders the enzyme an even more attractive drug target.147 Nevertheless, only a limited number of inhibitors have been characterized to date. As for IspD and IspE, these include pyrimidine derivatives, in this case small molecules of the thiazolopyrimidine class.165 More recently, symmetrical aryl bis-sulfonamides were shown to inhibit A. thaliana and P. falciparum IspF at low micromolar to nanomolar concentrations.166 Several cocrystal structures of IspF in complex with a range of potential inhibitors showed the expected binding interactions between cytosine analogues and the substrate binding pocket.167 A fragment screening approach identified ligand interactions with other active-site pockets, as well as with a site outside the catalytic center.168 Inhibitor design targeting methylerythritol phosphate isoprenoid biosynthesis has also addressed the pathway’s final two enzymes, IspG and IspH. Because of their identical cofactor requirements and the shared structural features of their ligands, the two catalysts share certain requirements and constraints with respect to their inhibition. As a result, numerous compounds are active against both proteins, which might limit the development of resistance mechanisms as described above. In addition, the accumulation of HMBPP, which activates human T cells, is reduced, and undesired side effects on the host immune system might be avoided.118,169 Alkyne (in particular, propargyl) diphosphates belong to the most extensively tested groups of IspG/IspH inhibitors. For IspH, these molecules are thought to form π complexes with the [4Fe4S] cluster’s apical iron, an interaction that was also proposed to occur with the natural HMBPP substrate after initial binding and reorientation of its hydroxy moiety (see also section 2.8.3).117,170 Propargyl diphosphate, for example, was the first micromolar inhibitor to be discovered for the enzyme.117 Another promising rational approach is the replacement of the aforementioned hydroxy group with a thiol or amino moiety. The resulting compounds provided inhibition constants in the low nanomolar range and bind to IspH in a reversible competitive manner, analogously to its HMBPP substrate.171,172 They are, however, not converted by the enzyme. Similarly to IspH, alkyne diphosphates were repeatedly shown to inhibit IspG.112,118,170 X-ray structures of the protein in complex with propargyl compounds suggest varying types of inhibitory effects, including competitive binding (here resembling HMBPP as a product) or steric clashes and displacement of the cofactor’s apical iron.111 A common feature of the most successful IspG and IspH

unique to the MEP catalyst is crucial. Alternatively, ligands with less promiscuous binding properties or those that form specific interactions with allosteric pockets can target enzymes highly selectively.147 Since its discovery, attempts have been made to identify inhibitors for all members of the MEP pathway. The diverse approaches ranged from the rational design of substrate mimics to the high-throughput screening (HTS) of large compound libraries. Deoxyxylulose phosphate synthase (DXS) constitutes the first enzyme of the pathway and, together with IspC, provides the highest flux-control coefficients for downstream reactions. In addition, the enzyme’s double functionality in vitamin B1 and vitamin B6 biosynthesis, as well as its proposed regulatory function, render it an even more attractive drug target.50,147,150 Nonetheless, comparatively little is known about inhibition of the synthase. Unlike other pathway constituents, no crystal structures are currently available for DXS in complex with either natural or synthetic ligands, aside from its ThDP cofactor, which makes rational drug design more challenging. The enzyme was, however, found to be inhibited by ketoclomazone, a derivative of the commercial herbicide clomazone.151,152 Kinetic analyses with DXS from Haemophilus inf luenzae suggested that the small molecule interacts with its protein target in a substrate-like manner and displays noncompetitive and mixed inhibition with respect to pyruvate and GAP binding, respectively.151 The best-studied drug target of the MEP pathway to date is its second catalyst, IspC. Homologues of the enzyme from bacteria, plants, and the apicomplexan Plasmodium species are inhibited by fosmidomycin, a small molecule resembling the natural rearrangement product.153−155 Whereas the inhibitor’s structure and its antimicrobial properties were known by 1980, its interaction with IspC, binding mode and potential as a drug, especially against P. falciparum as the causative agent of malaria, were elucidated two decades later.60,153,156 Steady-state kinetic analyses revealed a noncompetitive inhibition of cofactor (NADPH) binding, followed by slow, tight binding of fosmidomycin itself, resulting in the competitive inhibition of DXP substrate turnover.157 A ternary structure of IspC in complex with Mn2+ and fosmidomycin subsequently elucidated the inhibitor’s binding mode, which is thought to differ from the natural substrate.58,60 Highly promising findings by Jomaa and co-workers showed that mice infected with Plasmodium vinckei, the rodent equivalent of P. falciparum, were cured after treatment with fosmidomycin. Furthermore, the in vitro growth inhibition of the multidrug-resistant human pathogen was achieved at submicromolar concentrations.153 In subsequent years, however, clinical studies revealed high rates of recrudescent infections in malaria patients, as a consequence of which fosmidomycin was tested in combinatorial therapy with the lincosamide antibiotic clindamycin.158 IspD catalyzes the unusual cytidilation of MEP to CDP-ME and has been the target of HTS approaches to find efficient, small-molecule inhibitors of nonmevalonate biosynthesis. Two of the most significant sets of hits in these screens belong to the group of azolopyrimidines and the highly halogenated pseudilin natural products.159,160 Representatives of both inhibitor groups have been cocrystallized with IspD from A. thaliana, where they bind in allosteric pockets. Among other features, conformational changes and steric clashes in the active site prevent substrate binding and explain these molecules’ inhibitory effect in the nanomolar and low micromolar ranges. Although these are promising leads against plant and apicomplexan IspD 5696


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structures and mechanisms of all seven enzymes involved in the MEP pathway. Nonetheless, many of their biotechnological and pharmacological applications remain unexploited, leaving exciting future developments in this field to be expected.

inhibitors is the presence of a terminal diphosphate group. Although this moiety ensures efficient binding in a positively charged pocket of the enzymes’ active site, it significantly reduces cellular uptake and consequently the bioavailability of potential drugs. Efforts to replace the diphosphate with other functional groups obliterates the compounds’ inhibitory potential.118,149 In summary, each catalyst of the MEP pathway constitutes a valuable drug target with the potential to selectively inhibit crucial metabolic reactions in many notorious pathogens. Diverse approaches ranging from high-throughput screening to rational design based on crystallographic and biophysical data have provided promising leads, including a clinical-trial stage drug. Nonetheless, future efforts are required to address limitations such as cytotoxicity, side reactions, and the limited bioavailability of the currently available inhibitors.

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Michael Groll: 0000-0002-1660-340X Author Contributions

The manuscript was written through contributions of all authors. Notes

4. CONCLUSIONS AND OUTLOOK Isoprenoids constitute one of the largest classes of natural compounds. For many years, a biosynthetic pathway based on mevalonate as its key intermediate was seen as the exclusive metabolic route to IPP and DMAPP, the universal building blocks in all of these compounds. With the advent of whole genome sequencing, however, a species-specific second pathway emerged in which 2-C-methyl-D-erythritol-4-phosphate (MEP) is synthesized in the first committed step. In the period of a few years, most of its key catalysts were discovered and characterized in quick succession. The new pathway revealed a number of unusual and even unprecedented enzymatic reactions, including the IspDmediated cytidylation of MEP or the two single-electron reduction and concomitant hydroxy-group elimination reactions catalyzed by the iron−sulfur enzymes IspG and IspH. This use of a reducing metal cofactor in particular is characteristic for “ancient” enzymes, and the existence of two parallel pathways leading to the same isoprenoid building blocks raises interesting questions with respect to their evolutionary origin, age, and early functions. With the exception of few bacterial species and plants, which use both the mevalonate and MEP pathways in a compartmentalized manner and for the production of distinct downstream metabolites, isoprenoid biosynthesis has developed in a species-specific manner. Whereas mammalian cells rely entirely on the mevalonate pathway, many bacterial and apicomplexan species exclusively use the MEP pathway. These include a number of notorious and often multidrug-resistant pathogens such as P. falciparum or M. tuberculosis. Such a species distribution and selectivity renders their enzymes promising drug targets in the fight against many widespread infectious diseases. In addition to their pharmacological relevance, isoprenoids are essential in natural product biosynthesis and, consequently, important for diverse biotechnological applications. These range from the extraction or synthesis of natural aroma and flavor compounds, to the use of prenylation in protein immobilization or bioorthogonal modifications and the largescale production of medically relevant isoprenoids, especially di- and triterpenoids.173,174 As a result, the metabolic engineering of isoprenoid biosynthesis has attracted increasing attention in recent years, and the MEP pathway, which is already inherent to a range of bacterial and plant host systems, is an ideal target for this purpose. Intense research efforts of the past two decades have answered many questions regarding the three-dimensional

The authors declare no competing financial interest. Biographies Annika Frank obtained her B.Sc. in Biology in 2009 from the University of Applied Sciences in Rheinbach, Germany, and the University of Aberdeen (Scotland), where she worked on the genetics of Streptomyces coelicolor bacteriophages under Professor Maggie Smith. She subsequently carried out her Ph.D. studies with Professor Gideon Grogan at the University of York (England), where she analyzed the structures and functions of carbon−carbon bond hydrolases and lyases for biocatalytic applications. Since 2014, she has been a postdoctoral fellow with Professor Michael Groll at the Technische Universität München (Germany), where she has been working on a variety of biocatalytically relevant enzymes, including members of the methylerythritol phosphate isoprenoid biosynthetic pathway. Her research interests focus on the structure, function, and engineering of enzymes for biotechnological applications. Michael Groll studied chemistry and received his Ph.D. for crystallographic and biochemical studies on the yeast 20S proteasome. After postdoctoral studies in the groups of Profs. Huber (Munich), Finley (Boston), Neupert (Munich), and Kloetzel (Berlin) he became assistant professor at the Charité (Berlin). Since 2007, he has been full professor of Biochemistry at the Technische Universität München. His major research interests focus on the structural and functional characterization of multiprotein complexes and the analysis of interactions between enzymes and their ligands, as well as their catalytic mechanisms.

ACKNOWLEDGMENTS This work is dedicated to Professor Adelbert Bacher on the occasion of his 75th birthday. We thank Dr. Camille le Chapelain and Dr. Robert Byrne for helpful discussions and careful proofreading and editing of this manuscript. We are grateful to the Deutsche Forschungsgemeinschaft (DFG Grant GR 1861/5-2) for financial support of our work. ABBREVIATIONS ADP adenosine diphosphate ATP adenosine triphosphate CDP-ME 4-diphosphocytidyl-2-C-methylerythritol CDP-MEP 4-diphosphocytidyl-2-C-methyl- D -erythritol 2phosphate CMP cytidine monophosphate CoA coenzyme A CTP cytidine triphosphate 5697


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DHAP DMAPP DXP DXR DXS EPR FPP GAP gcpE GHMP

dihydroxyacetone phosphate dimethylallyl diphosphate 1-deoxyxylulose 5-phosphate 1-deoxyxylulose 5-phosphate reductoisomerase deoxyxylulose phosphate synthase electron paramagnetic resonance spectroscopy farnesyl pyrophosphate glyceraldehyde-3-phosphate gene coding for protein E galacto-, homoserine-, mevalonate, and phosphomevalonate kinases GPP geranyl pyrophosphate GTP guanosine triphosphate HiPIP high-potential iron−sulfur protein HMBPP 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate HMG-CoA (S)-3-hydroxy-3-methylglutaryl-CoA HMGR (S)-3-hydroxy-3-methylglutaryl-CoA reductase HPLC high-performance liquid chromatography HTS high-throughput screening IET internal electron transfer IPI isopentenyl diphosphate isomerase IPK isopentenyl phosphate kinase IPP isopentenyl diphosphate KIE kinetic isotope effect LThDP lactylthiamin diphosphate MDD mevalonate diphosphate decarboxylase MDP mevalonate diphosphate ME 2-C-methyl-D-erythritol MEcP 2-C-methyl-D-erythritol-3,4-cyclophosphate MEcPP 2-C-methyl-D-erythritol-2,4-cyclodiphosphate MEP 2-C-methyl-D-erythritol-4-phosphate MK mevalonate kinase MS mass spectrometry MVA (3R)-3,5-dihydroxy-3-methylpentanoic acid (mevalonate) MVAPD phosphomevalonate decarboxylase NAD+ nicotinamide adenine dinucleotide (oxidized) NADH nicotinamide adenine dinucleotide (reduced) NADP+ nicotinamide adenine dinucleotide phosphate (oxidized) NADPH nicotinamide adenine dinucleotide phosphate (reduced) NMR nuclear magnetic resonance ORF open reading frame PDHC pyruvate dehydrogenase complex PDB Protein Data Bank PDB ID Protein Data Bank identifier P-loop phosphate-binding loop PM phosphomevalonate PMK phosphomevalonate kinase PP pyrophosphate PYR (amino-)pyrimidine ThDP thiamin diphosphate TLC thin-layer chromatography UTP uridine triphosphate UV−vis ultraviolet−visible

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