Substrate Specificity and Engineering of Mevalonate 5-Phosphate

Article

Substrate specificity is published by the American Chemical Society. 1155 and engineering Sixteenth Street N.W., Washington, DC 20036

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of mevalonate 5phosphate decarboxylase

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Subscriber access provided by UNIV OF Published by American SOUTHERN INDIANA Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to

Suzanne T Thomas, Gordon V Louie, Johnathan W Lubin, Victoria Lundblad, and Joseph Noel is published P. by the American Chemical Society. 1155

ACS Chem. Biol.,Sixteenth Just Accepted Street N.W., Manuscript • DOI: 10.1021/ Washington, DC 20036

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Just Accepted

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Page 1 of 42

O O ACS Chemical OH Biology O

HO

P O O-

mevalonate 5-phosphate (MVAP)

1 2 3 4 5 6 7 8

MVA O HO

ATP ADP OH

PMK

MPD

O O P P O - O - OO O

ATP ADP+CO2 O

O P O O-

mevalonate 5-diphosphate (MVAPP) isopentenyl phosphate (IP) ATP ADP+CO2

MDD

IPK

ATP ADP


AltMVA1

ACS Paragon Plus Environment isopentenyl pyrophosphate (IPP)

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 42

Substrate specificity and engineering of mevalonate 5-phosphate decarboxylase

Suzanne T. Thomas1,2,*, Gordon V. Louie1,2, Johnathan W. Lubin 1,3, Victoria Lundblad1,3, Joseph P. Noel1,2,* 1

Salk Institute for Biological Studies, 2 Howard Hughes Medical Institute, 3Division of

Biological Sciences, University of California San Diego *To whom correspondence should be addressed: [email protected]

O

OH O

HO

O P O O-

mevalonate 5-phosphate (MVAP)

MVA O HO

ATP ADP

OH

PMK

MPD

ATP ADP+CO 2


O

mevalonate 5-diphosphate (MVAPP) ATP

ADP+CO 2

MDD

O P O O-

isopentenyl phosphate (IP) IPK

ATP ADP


AltMVA1

isopentenyl pyrophosphate (IPP)

ABSTRACT A bifurcation of the mevalonate (MVA) pathway was recently discovered in bacteria of the Chloroflexi phylum. In this alternative route for the biosynthesis of isopentenylpyrophosphate (IPP), the penultimate step is the decarboxylation of (R)mevalonate 5-phosphate ((R)-MVAP) to isopentenyl phosphate (IP), which is followed by the ATP-dependent phosphorylation of IP to IPP catalyzed by isopentenyl phosphate kinase (IPK). Notably, the decarboxylation reaction is catalyzed by mevalonate 5-phosphate decarboxylase (MPD), which shares considerable sequence similarity with mevalonate diphosphate decarboxylase (MDD) of the classical MVA pathway. We show that an enzyme originally annotated as an MDD from the Chloroflexi bacterium Anaerolinea thermophila

ACS Paragon Plus Environment

1

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ACS Chemical Biology

possesses equal catalytic efficiency for (R)-MVAP and (R)-mevalonate 5-diphosphate ((R)MVAPP). Further, the molecular basis for this dual specificity is revealed by near atomicresolution X-ray crystal structures of A. thermophila MPD/MDD bound to (R)-MVAP or (R)MVAPP. These findings, when combined with sequence and structural comparisons of this bacterial enzyme, functional MDDs, and several putative MPDs, delineate key active-site residues that confer substrate specificity and functionally distinguish MPD and MDD enzyme-classes. Extensive sequence analyses identified functional MPDs in the halobacteria class of archaea that had been annotated as MDDs. Finally, no eukaryotic MPD candidates were identified, suggesting the absence of the alternative MVA (altMVA) pathway in all eukaryotes, including, paradoxically plants, which universally encode a structural and functional homolog of IPK. Additionally, we have developed a viable engineered strain of Saccharomyces cerevisiae as an in vivo metabolic model and a synthetic biology platform for enzyme engineering and terpene biosynthesis in which the classical MVA pathway has been replaced with the altMVA pathway. INTRODUCTION Isopentenyl diphosphate (IPP) is the ubiquitous precursor of a sizable group of functionally and structurally diverse natural products, the terpenes. Terpenes play critical roles in numerous biological processes, including growth, development, and defense.1 IPP is therefore an essential metabolite in all three domains of life, Archaea, Bacteria, and Eukarya. This 5-carbon building block typically originates from either of two independent biosynthetic routes, the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways.


2


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The phylogenetic distribution of the MVA and MEP pathways is complex. Most Eukarya utilize the MVA pathway with an unusual compartmentalization extending from the cytosol to include, in some cases, mitochondria and peroxisomes. Among eukaryotes, organisms of the plant kingdom are unique in employing the MVA pathway and also a plastid-localized MEP pathway, both of which are essential for plant survival. The majority of Bacteria utilize the MEP pathway exclusively, with notable exceptions including scattered species of Actinobacteria, Bacteroidetes, Chloroflexi, Firmucutes, and Proteobacteria phyla.2 In these latter examples, partial or complete MVA pathways appear to be present.2,3 Finally, most Archaea utilize some form of the MVA pathway, with species in the genera Sulfolobus, Metallosphaera, and Acidianus encoding a complete, classical MVA pathway.4-6 However, the majority of Archaea lack the penultimate enzyme, (R)-mevalonate 5-phosphate kinase, alternatively phosphomevalonate kinase (PMK), or both PMK and the ultimate enzyme, (R)mevalonate

5-diphosphate

decarboxylase,

alternatively

mevalonate

diphosphate

decarboxylase (MDD), of the MVA pathway. This unexpected absence of essential MVA pathway enzymes provided impetus for the discovery of additional catalytic diversity associated with non-canonical MVA pathways of IPP biosynthesis (Figure 1).


3

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O CoA S acetyl-CoA

+

O

O

CoA S acetoacetyl-CoA H2O

HMGS

CoASH OH O

O

CoA S HO 3-hydroxy-3-methylglutaryl-CoA 2 NADPH

HMGR O

OO P OO O

2 NADP+CoASH OH

M3K

OH HO mevalonate (MVA)

ATP ADP

OH HO mevalonate 3-phosphate

ATP

ATP MVK

O

ADP OH

HO

MVA

ADP

O PMD P O -O O

O

OH

PMK O O P P O - O - OO O

M3P5K

AltMVA3

mevalonate 5-phosphate (MVAP) ATP

ATP

O P O - OHO O trans-anhydromevalonate 5-phosphate O

MPD

CO2

AHMPD

ADP+CO2

ADP+CO2

MDD

IPK

ADP

OO P OO O

O P O - OHO O mevalonate 3,5-bisphosphate

MBD

O P HO AltMVA1 O - OO mevalonate 5-diphosphate (MVAPP) isopentenyl phosphate (IP) ATP

AltMVA2

CO2 + PO4-3

ATP ADP

O O P P O - O - OO O isopentenyl pyrophosphate (IPP)

Figure 1. Isopentenyl diphosphate synthesis via the classic mevalonate and alternative mevalonate pathways. Enzymes in green are specific for the classic mevalonate pathway. Enzymes in pink, blue and green are specific to the altMVA1, altMVA2, and altMVA3 pathways respectively. Enzymes in black are shared by more than one pathway. HMGS, 3hydroxy-3-methylglutary-CoA synthase; HMGR, 3-hydroxy-3- methylglutary-CoA reductase, MVK, mevalonate kinase, PMK, phosphomevalonate kinase, MDD, mevalonate-5diphosphate decarboxylase, MPD, mevalonate 5-phosphate decarboxylase, IPK, isopentenyl


4


Page 6 of 42

kinase; PMD, mevalonate 5-phosphate dehydratase; AHMPD, trans-anhydromevalonate 5phosphate decarboxylase; M3K, mevalonate-3-kinase; M3P5K, mevalonate-3-phosphate-5kinase; MBD, mevalonate-3,5-diphosphate decarboxylase. The classical MVA pathway comprises five enzymatic reactions that convert two- and four-carbon building blocks provided by acetyl-CoA and acetoacetyl-CoA, respectively, into the 5-carbon IPP product. The first three steps of this pathway are common also to the alternative MVA (altMVA1) pathway. In contrast, the final two transformations, phosphorylation of (R)-MVAP and decarboxylation of (R)-MVAPP, catalyzed sequentially by PMK and MDD in the classical MVA pathway, occur in “reverse chemical” order in the altMVA1 pathway (Figure 1). The penultimate step of the altMVA1 pathway is the decarboxylation of (R)-MVAP catalyzed by (R)-MVAP decarboxylase (MPD), yielding isopentenyl phosphate (IP). MPD was first identified in the Chloroflexi bacterium Roseiflexus castenholzii, encoded by a gene initially annotated as encoding a classical MDD.7 The ultimate step of all altMVA pathways, phosphorylation of IP, is catalyzed by isopentenyl phosphate kinase (IPK). IPK activity and the associated enzyme were first functionally and structurally characterized in Archaea,8,9 and subsequently, IPK was found throughout the plant kingdom.7 More recently, the archaeal halobacterium Haloferax volcanii, which utilizes the altMVA1 pathway for IPP biosynthesis, was shown to encode IPK and MPD.10 New reports describe another deviation in the alternative MVA pathway (altMVA2) occurring in the archaeal class Thermoplasmata (Figure 1). The altMVA2 pathway utilizes the first two reactions of the MVA and altMVA1 pathways, catalyzed by 3-hydroxy-3methylglutary-CoA synthase (HMGS) and 3-hydroxy-3- methylglutary-CoA reductase (HMGR), and also the final reaction of the altMVA pathways, catalyzed by IPK. Additionally, two central reactions seemingly unique to the altMVA2 pathway are catalyzed by more


5

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divergent homologs of MDD/MPD, (R)-mevalonate-3-kinase (M3K) and (R)-mevalonate-3,5diphosphate decarboxylase (MBD).11,12 This pathway is speculated to have evolved in extremophiles that survive in low pH environments.13 The variations in the biosynthetic routes utilized in the MVA, altMVA1, and altMVA2 pathways, and additionally a third altMVA3 pathway14 (Figure 1) arise largely from distinct activities of MVK and MDD/MPD enzymes, which are all members of the galacto- (EC:2.7.1.6), homoserine (EC:2.7.1.39), mevalonate

(EC:2.7.1.36)

and

phosphomevalonate

(EC:2.7.4.2)

kinase

(GHMP)

superfamily.15 It has become increasingly evident that the detailed characterization of a presumed altMVA pathway requires careful functional annotation of the MDD/MPD enzymes involved. IPK, a member of the amino acid kinase (AK) family,16 catalyzes the ultimate step of the altMVA pathways, but its distribution is enigmatic. Plausibly, in any organism encoding a gene for IPK, an altMVA pathway may be operative. However, although functional IPK orthologues have been found in every sequenced green plant genome, plants universally employ both the MVA and MEP pathways7 but not an altMVA pathway.17,18 Furthermore, annotated plant MDDs behave in a canonical fashion and are unable to catalyze in vitro the MPD reaction, the decarboxylation of (R)-MVAP to IP.17 Similarly, the archaea Sulfolobus solfataricus possesses an IPK, but also a complete classical MVA pathway and canonical MDD.4 Thus, in both green plants and archaea like Sulfolobus solfataricus, important new questions are raised concerning IPK's biological role(s) in the terpenoid metabolism. The latest data establish IPK in plants as an important member of the isoprenoid biosynthetic network, acting in the regulation of compartmentalized MVA and MEP pathway-derived terpene production, but the full scope of IPK’s roles remains unresolved.


6


Page 8 of 42

Here we identify an annotated MDD from the Chloroflexi bacterium Anaerolinea thermophila with equivalent specific activities toward (R)-MVAP and (R)-MVAPP, and describe protein x-ray crystal structures of substrate–bound complexes of this bifunctional A. thermophila enzyme. To complement these discoveries, we report the first plant MDD protein crystal structure from Arabidopsis thaliana, AtMDD1. AtMDD1 is highly specific for (R)-MVAPP as substrate.17 Using amino-acid sequence and three-dimensional structural comparisons amongst the MDD/MPD family, we identify comprehensively the contributions of key active-site residues to the substrate specificity-permissiveness properties of this enzyme family. This combination of structural information is then utilized to predict the distribution of the altMVA pathways across the three domains of life. Finally, we demonstrate that heterologous expression of altMVA1 pathway genes, mpd and ipk from A. thermophila, can substitute for the classical MVA pathway in S. cerevisiae, setting the stage for quantifying the metabolic costs-benefits of operational classical and altMVA pathways in a single eukaryotic host. RESULTS and DISCUSSION Functional Characterization of Chloroflexi MVA Pathway Enzymes.

To identify

functional MPDs, we first focused on enzymes annotated as MDDs from two sequenced bacterial species of the Chloroflexi phylum, Anaerolinea thermophila and Herpetosiphon aurantiacus.19,20 Each protein was heterologously produced in E. coli using codon-optimized expression constructs, purified to homogeneity, and kinetically characterized using (R)MVAP or (R)-MVAPP and the co-substrate ATP using a pyruvate kinase-lactate dehydrogenase coupled assay.21 The formation of the appropriate decarboxylated product was confirmed by reversed-phase liquid chromatography (RPLC)–mass spectrometry (MS)


7

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(Supplementary Figure 1 and Supplementary Figure 2). Both the A. thermophila and H. aurantiacus enzymes displayed measurable activity for the conversion of (R)-MVAP to IP (Table 1). The H. aurantiacus enzyme behaved as a bona fide MPD, exhibiting the kinetic constants KMMVAP = 36 ± 5 M, KMATP = 325 ± 39 M, and kcat = 4.1 ± 0.1 s-1 with (R)-MVAP as substrate, but no measurable activity with (R)-MVAPP as substrate. The A. thermophila enzyme similarly converted (R)-MVAP to IP with kinetic constants KMMVAP= 29 ± 5 M, KMATP= 349 ± 91 M, and kcat = 1.6 ± 0.1 s-1, but surprisingly, also catalyzed the conversion of (R)MVAPP to IPP with kinetic constants KMMVAPP = 46 ± 6 M and kcat = 0.74 ± 0.04 s-1. Table 1. Steady-state kinetic constants for Chloroflexi mevalonate 5-phosphate decarboxylases and enzymes of Anaerolinea thermophila altMVA1 pathway. Organism

Enzyme

Roseiflexus Castenholzii

MPDc

Herpetosiphon aurantiacus

MPD

MPD Anaerolinea thermophila

MVK IPK

Substrate

kcat (s-1)

KMMVAP(P) (μM)a

KMATP (M)b

kcat/KMMVAP(P) (s-1M-1)

(R)-MVAP

1.7 ± 0.1

152 ± 38

190 ± 60

0.011 (± 0.003)

(R)-MVAPP

NA

NA

NA

NA

(R)-MVAP

4.1 ± 0.1

36 ± 5

349 ± 91

1.1 × 105 ± 0.2

(R)-MVAPP

NA

NA

NA

NA

(R)-MVAP

1.6 ± 0.1

29 ± 5

325 ± 39

6 × 104 ± 1

46 ± 6

ND

1.6 × 104 ± 0.2

(R)-MVAPP 0.74 ± 0.04 (R)-MVA

2.1 ± 0.1

44 ± 7

78 ± 33

4.7 × 104 ± 0.8

(R)-MVAP

NA

NA

NA

NA

IP

3.1 ± 0.1

34 ± 5

29 ± 14

9.4 × 104 ± 1.4

NA: no activity detected ND: not determined a: Apparent KM of (R)-MVAP, (R)-MVAPP, or (R)-MVA determined at 4 mM ATP. b: Apparent KM of ATP at 0.5 mM (R)-MVAP or 0.5 mM (R)-MVA. c: reported previously7

A. thermophila mevalonate decarboxylase was thus shown to be substrate ambiguous, at least in vitro, utilizing (R)-MVAP and (R)-MVAPP with almost equal catalytic efficiency. (R)-MVAPP is the penultimate metabolite of the classical MVA pathway and a product of PMK (Figure 1), an enzyme not found in bacteria of the Chloroflexi phyla, including A. thermophila.


8


Page 10 of 42

We next explored the possibility that A. thermophilia MVK (AthermMVK), a member of the same GHMP superfamily as PMK, possesses dual specificities and can phosphorylate (R)MVAP to supply (R)-MVAPP. AthermMVK was expressed in E. coli, purified to homogeneity, and assayed for ATP-dependent kinase activity with the presumptive substrates (R)-MVA or (R)-MVAP. AthermMVK catalyzed the phosphorylation of (R)-MVA to (R)-MVAP with kinetic constants of KMMVA = 44 ± 7 M, KMATP = 78 ± 33 M, and kcat = 2.1 ± 0.1 s-1 (Table 1). In contrast, no (R)-MVAPP production was detectible by RPLC-MS for AthermMVK using (R)MVAP as a substrate (Supplementary Figure 3). In order to verify the existence of a functionally complete altMVA pathway in A. thermophilia, we characterized the enzyme catalyzing the ultimate reaction, IPK (AthermIPK). Heterologously expressed and purified AthermIPK efficiently catalyzed the in vitro ATP-dependent phosphorylation of IP to IPP with kinetic parameters of KMIP = 34 ± 5 M, KMATP = 29 ± 14 M, and kcat = 3.1 ± 0.1 s-1 (Table 1). In A. thermophilia, the absence of a known source of (R)-MVAPP and the presence instead of a functional IPK suggest that the observed in vitro activity of mevalonate decarboxylase with (R)-MVAPP is unlikely to be of metabolic consequence. Instead, this enzyme ostensibly functions in vivo as an MPD, a component of the altMVA1 pathway as defined in other Chloroflexi bacteria.7 Further, the observed substrate permissiveness of this A. thermophilia MDD/MPD is consistent with a lack of selection pressure for increased substrate selectivity. We will refer herein to the annotated MDDs from A. thermophila and also H. aurantiacus as MPDs, AthermMPD and HaMPD, respectively. Three-dimensional architecture of AthermMPD. Single, diffraction-quality crystals of AthermMPD were obtained in the presence of (R)-MVAP or (R)-MVAPP, whereas


9

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crystallization trials with apo protein or protein with only the co-substrate ATP were unsuccessful. AthermMPD crystals belonged to space-group P21 and an initial structure solution was obtained by molecular replacement using a search model derived from Staphylococcus aureus MDD (SaMDD). SaMDD shares 39% amino-acid sequence identity with AthermMPD (Figure 2A). AthermMPD, like MDDs, belongs to the GHMP superfamily of ATP-dependent kinases. Refined structures were obtained for AthermMPD in complex with (R)-MVAP at 1.60 Å resolution (Rwork= 0.1710 and Rfree=0.1939) and with (R)-MVAPP at 2.20 Å resolution (Rwork= 0.1661 and Rfree=0.2061). AthermMPD and SaMDD (pdb 2HK2) superimposed with a root-mean-square deviation (rmsd) of 1.27 Å for the Cα atoms of 315 aligned residues out of a total of 322 residues in AthermMPD.


10


Page 12 of 42

A

AthermMPD AthalMDD

AthermMPD MVAP

B

C

D280

D280

R143 W18

R143

3.7

S140

S140

W18

Y17

Y17 S106

H194

S106 H194

S138 S190

S190 N20

N20

T191

R71

T191

R71

R26

S138

R26

AthermMPD MVAPP

D

D280

E

R143

D280 R143

S140

W18 Y17

W18 Y17

S106

H194

S138

N20

S190

A

R26

S106

H194

B

S190

S140

B S138

A N20

R71

R26

R71

T191

T191

AthermMPD N20K/H194M MVAPP

F

G

D280

D280 R143

R143 S140 W18

S140

W18 Y17

Y17 H194M

S106

H194M

S106

S190

S190 N20K

S138

N20K

S138

R71

R71 T191

R26

T191

R26

AthalMDD MVAPP

H

I

D310 R166

D310 R166

S163

W24

W24

S163 Y23 S216

S161 M220

S161 M220

K26 S129

S129

K26 T217

T217

R78


R78

11

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Figure 2. X-ray crystal structures of AthermMPD and AthalMDD. (A) Overlaid cartoon representations of AthermMPD dimer with (R)-MVAP bound (pink) and AthalMDD dimer with (R)-MVAPP bound (cyan). (B) Active site of AthermMPD with 2mFo-DFc difference density for (R)- MVAP (C) Active site of AthermMPD with MVAP binding interactions shown. (D) Active site of AthermMPD with 2mFo-DFc difference density for (R)- MVAPP bound in two conformations (E) Active site of AthermMPD with MVAPP binding interactions shown. (F) Active site of AthermMPD N20K/H194M with 2mFo-DFc difference density for (R)MVAPP and mutated residues (G) Active site of AthermMPD N20K/H194M with MVAPP binding interactions shown (H) Active site of AthalMDD with 2mFo-DFc difference density for (R)- MVAPP (I) Active site of AthalMDD with MVAPP binding interactions shown. Atoms colored by atom type: oxygen, red; nitrogen, blue; phosphorus, orange; sulfur, yellow. The asymmetric unit of AthermMPD crystals contains six protein molecules. These six independent copies form three equivalent homodimers (Figure 2A). The dimeric state of AthermMPD in solution was confirmed by analytical ultracentrifugation, from which a global fit of sedimentation-equilibrium profiles indicated an oligomer mass of 68 kDa consistent with two molecules of AthermMPD (71.4 kDa). The homodimeric arrangement observed for AthermMPD occurs in all structurally characterized MDDs,22-28 with the exception of monomeric Trypanosoma brucei MDD26 and Enterococcus faecalis MDD, 29 despite the rather limited area buried across the dimer interface (in the AthermMPD homodimer, ~1,100 Å2 corresponding to only 8% of the total available monomeric surface area). Role of AthermMPD Active-Site Residues in Substrate Recognition and Catalysis. Initial difference maps for (R)-MVAP or (R)-MVAPP co-crystallized samples showed strong, continuous electron density in the active-site pocket of each of the six AthermMPD molecules in the asymmetric unit. However, in co-crystallization trials of AthermMPD with (R)-MVAP or (R)-MVAPP and including the reaction product ADP, no additional residual electron density was observed in the nucleotide-binding pocket. (R)-MVAPP (pdb ligand ID DP6) or (R)-MVAP (ligand ID PMV) were unambiguously modeled and included in the refinement of each AthermMPD-substrate complex (Figure 2B and 2D). The (R)-MVAP and (R)-MVAPP


12


ligands bound at full occupancy, as indicated from occupancy refinement and comparison of refined atomic displacement parameters with those of neighboring protein atoms. Superposition of the (R)-MVAP- and (R)-MVAPP-bound AthermMPD structures illustrates the common constituents of these two ligands (atoms of the (R)-mevalonate moiety and the α-phosphate group) are positioned identically. The β-phosphate group of (R)-MVAPP can occupy two distinct sites (Figure 2D); the predominant binding mode of (R)-MVAPP in AthermMPD (average occupancy of 0.58 for the six copies in the asymmetric unit) corresponds closely to the binding mode of this substrate observed in both Arabidopsis thaliana MDD (AthalMDD) (Figure 2H) and Staphylococcus epidermidis MDD (SepMDD, pdb 4DU7).25 Previous kinetic, structural and mutagenic studies have demonstrated that an Arg and an Asp residue (Arg143 and Asp280 of AthermMPD) are essential for decarboxylase activity.23,26,28,30 Multiple sequence alignments reveal that both the Arg and Asp residues are absolutely conserved in all MDDs, Chloroflexi MPDs, and the characterized M3K and MBD from Thermoplasmata (Figure 3, Supplementary Figure 4).

MDD

MPD

20 |

MBD M3K

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 42

A. thermophila H. aurantiacus C. aerophila Roseiflexus sp R. castenholzii Chloroflexus sp C. aurantiacus C. aggregans O. trichoides H. volcanii S. solfataricus S. epidermis H. sapiens A. thaliana S. cerevisiae F. johnsoniae T. acidophilium P. torridus T. acidophilium P. torridus

26 |

KYWGNR-DAVLRIP KYWGQH-DSQLTLP KYWGVA-DPHLNIP KYHGLS-DWKYRIA KYHGLS-DWDYRIA KYHGLS-DWTQRIA KYHGLS-DWTQRIA KYHGLS-DWVQRIA KYHGLS-DWHYRIA KYHGMR-DTERRMP KYWGKRGDERLNLP KYWGKA-DETYIIP KYWGKR-DEELVLP KYWGKR-DEVRILP KYWGKR-DTKLNLP KYWGKK-E—NQIP LLGGIA-NPVTRTP LLGGIS-DKKNRIP KFLGYY-DRENRIA KFLGYY-DRDNNIA

71 | EPA-LKRVSHFLD GRQ-FERVIQQIE GPK-AERVTRHLD GRE-LERVQQSLD RGRE-LERVRQSL GRE-LERVVTVLD GRE-LERVVTVLD GRE-LERIVHVLD GRD-LDRVVQTLN GRG-AERIQAVVD -DEMKEYAGRVLD AKE-KEKIQNYMN -VG-QPRLQACLR -LS-GSRYQNCLR -ID-NERTQNCLR -DF-KPKIQKFLE -DD-NRSVRRVLD LNS-DRSPSKVID -KY-YKKAKFALD -KY-YKRAEKALS

106 | GIASSAAA GIASSAAA GIASSASA GLGSSASA KGLGSSAA GLGTSASA GLGTSASA GLGTSASA GLGTSASA GFGSSASG GLASSAAG GLASSASA GLASSAAG GLASSAAG GLASSAAG GIASSASG LSGSSDAG LSGSSDSG GLSESSAV GLGESAAV

138 140 143 | | | GSGSACRSI GSGSACRSI GSGSASRSI LAGSGCRSA LAGSGCRSA LAGSGCRST LAGSGCRST LAGSGCRSA LAGSGCRSA GSASAARAV GSGSACRSM GSGSASRSI GSGSACRSL GSGSACRSL GSGSACRSL GSGSACRSV VSESAGRSL ISESVGRSL VSGSGTRAA VSGSGTRSA

190 194 | | PIGSTQGH-ALASTS HVASTSGH-SVATTS TVSSQNGH-ALALTS GLKTEQAH-MDAPAS GLKTEQAH-LDAPGS GLKTEQAH-HDAPNS GLKTEQAH-HDAPNS GLKTEQAH-HDAPQS GLKTESAH-HDAPES --ETEQAH-AEAADS KISSRKGMIRSAETS KVSSRSGMSLTRDTS LTGSTVGMRASVETS ETSSTSGMRESVETS DVSSTQGMQLTVATS QVSSTVGH-DLMHNH -NPSDVIH-QNIVRS -KPSNEIH-ENIIKH -VATDNAH-SIAVNS -IETLNAH-DYASSS

280 | AYTLDAGPNV YWTIDAGPNV YFTIDAGPNV YCSTDTGPTA YCSTDTGPTA YASTDTGPTV YASTDTGPTV YASTDTGPTV YASTDTGPTV YFSTDTGASV GYTFDAGPNP YFTMDAGPNV AYTFDAGPNA AYTFDAGPNA AYTFDAGPNA CFTLDAGANV AYIVTGGSNV SYIVTGGPNV YFTADTGPSI YFTSDTGTSI

Figure 3. Multiple sequence alignment of MPDs, MDDs, M3Ks, and MBDs discussed in the text. Numbers above the sequence refer to the amino acid number of AthermMPD. Alignment


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was generated by T-Coffee and visualized by BOXSHADE. Sequences include Anaerolinea thermophila MPD (E8N6F3), Herpetosiphon aurantiacus MPD (A9B5M2), Caldilinea aerophila MPD (I0I5R2), Roseiflexus species MPD (A5V173), Roseiflexus castenholzii MPD (A7NHV3), Chloroflexus species MPD (WP031458365), Chloroflexus aurantiacus MPD (A9WEU8), Chloroflexus aggregans MPD (B8G8V9), Oscillochloris trichoides MPD (E1IFM0), Haloferax volcanii MPD (D4GXZ3), Sulfolobus solfataricus MDD (Q97UL5), Staphylococcus epidermis MDD (W1WIY5), Homo sapiens MDD (P53602), Arabidopsis thaliana MDD1 (O23722), Saccharomyces cerevisiae MDD (P32377), Thermoplasma acidophilum M3K (Q9HIN1), Picrophilus torridus M3K (AAT43941), Thermoplasma acidophilum MBD (Q9HJS1), and Picrophilus torridus MBD (Q6L1T9). The full alignment can be found in the Supplementary Figure 4. In addition to these two key catalytic residues, several other polar side-chains lining the active-site pocket of AthermMPD form hydrogen-bonding interactions with the (R)MVAP or (R)-MVAPP substrates, primarily with the 5-phosphate or 5-diphosphate groups (Figure 2C, 2E). Aside from Arg26 and His194 discussed below, of particular prominence are three serine residues, Ser138, Ser140, and Ser190, which are highly conserved across MDDs and have previously been functionally characterized. 31 In the substrate-bound AthermMPD complexes, all three Ser residues are involved in hydrogen bonds with the single phosphate group of (R)-MVAP (Figures C). Although Ser138 is conserved in AthermMPD and HaMPD, it surprisingly is replaced by Ala in other Chloroflexi MPDs from the Roseiflexus and Chloroflexus genera (including the previously characterized R. castenholzii MPD7), and therefore appears to be nonessential for MPD activity (Figure 3). Ser138 and Ser140 in AthermMPD are involved in a binary interaction with a phosphate oxygen atom of (R)-MVAP, an arrangement that may mitigate the effect on (R)-MVAP binding of the Ala substitution at position 138 in the Roseflexus and Chloroflexus MPDs. Ser138 and Ser190 in AthermMPD also form hydrogen bonds with the -phosphate group of (R)-MVAPP, contributing to a network of interactions within two subsites that are distal to the binding pocket occupied by the smaller (R)-MVAP molecule (Figure 2E). In


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conformation A of (R)-MVAPP, the β-phosphate moiety participates in a number of polar interactions with Arg26, Arg71, Ser138, Thr191, and Tyr17. In conformation B, the βphosphate group forms hydrogen bonds with Ser106 and Ser190. Three-dimensional architecture of AthalMDD. Single, diffraction-quality crystals of AthalMDD were obtained in the presence (R)-MVAPP, and belong to space-group P41212. A structure of AthalMDD in complex with (R)-MVAPP was obtained initially by molecular replacement with a search model derived from human MDD (which shares 50% amino acid sequence identity with AthalMDD), and refined at 2.3 Å resolution (Rwork= 0.1907 and Rfree= 0.2273). The asymmetric unit of AthalMDD crystals contains one protein molecule, which forms the characteristic homodimeric arrangement with a crystallographic-symmetry related molecule (Figure 2A). AthalMDD has the archetypical GHMP α/β fold. AthalMDD and AthermMPD share 36% amino acid sequence identity, and superimpose structurally with a rmsd of 1.49 Å for the Cα atoms of 308 aligned residues out of 406 and 322 residues in AthalMDD and AthermMPD, respectively. Comparison of the (R)-MVAPP-bound structures of AthalMDD and AthermMPD shows that the (R)-MVAPP substrates are almost identically positioned in the respective active sites (Figure 2H). Furthermore, the amino-acid residues lining the active site pocket and interacting with the (R)-MVAPP are largely conserved, including the catalytic residues Arg166 and Asp310, and the conserved hydrogen-bonding side chains of Ser161, Ser163 and Ser216 (Figure 2I). Determinants of Substrate Specificity of AthermMPD and AthalMDD. Detailed sequence analyses combined with structural comparisons of (R)-MVAP-bound AthermMPD and (R)MVAPP-bound AthalMDD revealed two conspicuous amino-acid differences that likely


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underlie the distinct substrate preferences of the MPD and MDD decarboxylases. First, in the MDDs, a highly-conserved lysine (Lys26 in AthalMDD) is located at the base of the substratebinding pocket, where it forms an ion pair with the β-phosphate group of (R)-MVAPP (Figure 2I). In Chloroflexi and H. volcanii MPDs, the corresponding residue is variable in identity with an asparagine (Asn20) occurring in AthermMPD and valine, leucine, methionine, and glutamine occurring in other MPDs (Figure 3). The notable absence of lysine at this position in the MPDs is likely a primary contributing factor in the relatively poor binding of (R)MVAPP by this class of mevalonate decarboxylases. The second key determinant of substrate specificity is a histidine residue (His194 in AthermMPD) that is conserved exclusively in the MPDs from Chloroflexi and H. volcanii (Figure 3). In AthermMPD, the observed conformational (gauche¯/χ2=-70°) and tautomeric (Nε2-H neutral) states of the His194 imidazole side-chain are imposed by a hydrogen bond accepted by the His194-ND1 atom from the backbone amide nitrogen of Gly19, and sandwiching of the imidazole ring by the neighboring aromatic side chains of Tyr17 and Trp18. In the substrate-bound complexes of AthermMPD, the hydrogen-bond donor His194 Nε2-H forms a charged hydrogen bond with the phosphate group of (R)-MVAP (Figure 2C) or an uncharged hydrogen bond with the oxygen atom bridging the α and β phosphate groups of (R)-MVAPP in binding conformation A (Figure 2E). In all known MDDs, the residue corresponding to His194 is a conserved methionine (Met220 in AthalMDD) with a proposed structural role in positioning the side chain of the lysine side chain responsible for binding the -phosphate of (R)-MVAPP (Figure 2I).26 Such a function would be obviously unnecessary in the MPDs which lack this lysine.


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To further probe the role of these conserved lysine and histidine residues in the mevalonate decarboxylases, we generated N20K and H194M single mutants and the N20K/H194M double mutant of the substrate ambiguous AthermMPD and then assessed the mutational effects on enzyme activity with (R)-MVAP and (R)-MVAPP (Table 2). The AthermMPD N20K substitution caused a 10-fold increase in KM and a 23-fold decrease in kcat with (R)-MVAP, and less drastically, a 3-fold decrease in KM and 2-fold decrease in kcat with (R)-MVAPP. The severe impairment in activity observed for the N20K mutant with (R)-MVAP was unexpected given the sequence variability of the Asn20 site amongst Chloroflexi MPDs. Significantly, the introduction of Lys20 improved (R)-MVAPP binding by the AthermMPD mutant, consistent with the function of this lysine in forming a favorable ionic interaction to the β-phosphate group of (R)-MVAPP, as observed in substrate-bound complexes of both AthalMDD and the N20K/H194M variant of AthermMPD (see below). Table 2. Steady-state kinetic constants for AthermMPD mutants with (R)-MVAP or (R)MVAPP and ATP substrates. Anaerolinea thermophila WT

N20K

H194M N20K/ H194M R26V

R71A

Substrate

kcat (s-1)

KMMVAP(P) (μM)a, b

KMATP (M)b

kcat/KMMVAP(P) (s-1M-1)

(R)-MVAP

1.6 ± 0.1

29 ± 5

349 ± 91

6 × 104 ± 1

(R)-MVAPP

0.74 ± 0.04

47 ± 10

ND

1.6 × 104 ± 0.2

(R)-MVAP

0.07 ± 0.01

280 ± 80

ND

2.5 × 102 ± 0.8

(R)-MVAPP

0.40 ± 0.03

17 ± 5

207 ± 37

2.4 × 104 ± 0.7

(R)-MVAP

1.1 ± 0.1

700 ± 100

ND

1.5 × 103 ± 0.3

(R)-MVAPP

1.2 ± 0.07

232 ± 47

565 ± 57

5 × 103 ± 1

(R)-MVAP

NA

NA

NA

NA

(R)-MVAPP

0.38 ± 0.02

18 ± 3

202 ±- 43

2.1 × 104 ± 0.4

(R)-MVAP

1.66 ± 0.05

61 ± 6

1900 ± 650

2.7 × 104 ± 0.3

(R)-MVAPP

0.29 ± 0.08

4400 ± 1700

ND

6.6 × 102 ± 0.3

(R)-MVAP

0.022 ± 0.001

56 ± 13

~ 20 000

3. 9 × 102 ± 0.9

(R)-MVAPP

0.037 ± 0.002

179 ± 30

ND

2.0 × 102 ± 0.4


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NA: no activity detected ND: not determined a: Apparent KM of (R)-MVAP or (R)-MVAPP determined at 4 mM ATP. b: Apparent KM of ATP at 0.5 mM of (R)-MVAP or (R)-MVAPP.

The substitution of His194 by methionine in AthermMPD resulted in no significant change in kcat with either (R)-MVAP or (R)-MVAPP, but 24-fold and 5-fold increases in KMMVAP and KMMVAPP, respectively (Table 2). Notably, the substantially greater effect on (R)-MVAP binding emphasizes the particularly strong stabilizing contribution of the charged hydrogenbond between Nε2-H of the His194 imidazole side chain and the phosphate group of (R)MVAP in comparison to a weaker contribution from the neutral hydrogen bond between Nε2-H and the diphosphate-group bridging oxygen of (R)-MVAPP. In combination, the N20K/H194M dual substitution in AthermMPD abolished activity with (R)-MVAP as a substrate while conferring near wild-type activity with (R)-MVAPP, and thereby converted the substrate ambiguous AthermMPD to an enzyme with MDD activity exclusively (Table 2). Moreover, the crucial roles in the MDDs of the conserved lysine and methionine residues in excluding activity with (R)-MVAP are underscored. Such activity, in the context of the classical MVA pathway and the absence of IPK activity, would result in the nonproductive formation of the dead-end intermediate IP. Whereas strict selection against activity with (R)-MVAP is critical in the case of MDD, substrate ambiguity can be tolerated in the case of MPD, where the activity with (R)-MVAPP produces IPP, the intended ultimate product of the altMVA pathway. The MDDs exploit two mechanisms for discriminating against (R)-MVAP as substrate, both dependent on the role of the (R)-MVAP phosphate group in ionic interactions. First, the lysine side chain normally interacts with the β-phosphate group of (R)-MVAPP, and the formation of an interaction with the phosphate group of the shorter (R)-MVAP molecule would likely compromise the substrate-binding mode that


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promotes optimal catalytic activity as inferred from Figure2C and 2I. Second, the methionine in MDD contributes to an unfavorable environment for the single phosphate group of (R)MVAP, owing to the absence of a charged hydrogen-bond interaction for stabilization of the phosphate's negative charge. Under cocrystallization conditions established for wild-type AthermMPD in the presence of either (R)-MVAP or (R)-MVAPP substrates, crystals of the AthermMPD N20K/H194M variant were readily obtained with (R)-MVAPP but not with (R)-MVAP, consistent with the inactivity of this variant with (R)-MVAP. The structural model of the (R)MVAPP-bound N20K/H194M variant was refined at 1.95-Å resolution to final Rwork= 0.1879 and Rfree=0.2180. In the N20K/H194M structure, (R)-MVAPP is bound in a single conformation corresponding to conformation A described earlier for wild-type AthermMPD, with its β-phosphate group forming a salt-bridge hydrogen bonding interaction with the side chain of the mutant lysine residue (Figure 2F, 2G). Notably, the positioning of both (R)MVAPP and the key lysine residue observed here match closely those occurring in the AthalMDD-(R)-MVAPP complex. Furthermore, with wild-type AthermMPD, two binding modes are adopted by (R)-MVAPP, suggestive of a diminished binding specificity for this substrate; whereas, in marked contrast, the more selective N20K/H194M variant clearly disfavors the alternative binding conformation of (R)-MVAPP (Figure 2D). An additional feature of (R)-MVAPP binding common in MDDs is the involvement of an arginine residue in charge stabilization of the (R)-MVAPP β-phosphate group. Strikingly, the location of the arginine in the primary structure varies amongst MDDs from the three domains of life. In eukaryotic MDDs, this arginine (Arg78 in AthalMDD) is contributed by the N-terminal domain and is highly conserved. In bacterial MDDs, the arginine originates from


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the C-terminal domain, for example, Arg193 in SepMDD. Of the archaeal MDDs, few have been functionally or structurally characterized with the exception of Sulfolobus solfataricus which retains the arginine of bacterial MDDs, Arg195. 4,28 A distinct arginine residue (Arg26 in AthermMPD) from the N-terminal region occurs in the substrate-binding pocket of most Chloroflexi MPDs; and owing to its absence in bacterial, archaeal, or eukaryotic MDDs, we had previously proposed that this arginine may serve as a primary determinant for the preference of MPD for the substrate (R)-MVAP.7 However, our current structure determinations of substrate-bound complexes of AthermMPD show that the guanidinium group of Arg26 in fact forms a close ionic hydrogen bonding interaction with the β-phosphate group of (R)-MVAPP but not the single phosphate group of (R)-MVAP (Figure 2C, 2E). The involvement of AthermMPD Arg26 in conferring binding affinity for (R)-MVAPP was confirmed by kinetic analysis of an R26V mutant. This position is a valine in human MDD. In comparison to wild-type AthermMPD, the R26V mutant exhibited relatively unchanged KMMVAP and kcat values, but a markedly greater 90-fold increase in KMMVAPP (Table 2). Thus, the single R26V substitution successfully moved the substrate specificity strongly in favor of (R)-MVAP over (R)-MVAPP, with a 40-fold greater kcat/KM for (R)-MVAP compared to (R)-MVAPP. In addition, as described earlier, HaMPD was shown to be highly specific for (R)-MVAP with no detectable activity with (R)-MVAPP (Table 1), and accordingly also represents one of the Chloroflexi MPDs that bears a substitution, threonine, for the key arginine (Figure 3). We posit that any Chloroflexi MPD possessing this arginine is potentially active against (R)-MVAPP and like AthermMPD exhibits substrate ambiguity.


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The positional coincidence of the side-chain guanidinium groups of the nonhomologous Arg26 of AthermMPD, Arg78 of AthalMDD, and Arg193 of SepMDD exemplifies a remarkable example of evolutionary convergence in the utilization of an arginine in the recognition and binding of the -phosphate group of the (R)-MVAPP substrate (Figure 4). Notably, in the MPDs from the archaea H. volcanii and bacteria of the Chloroflexi phylum, the latter two of these arginine sites bear amino-acid substitutions or are involved in structural rearrangements, and consequently, these corresponding residues have alternative functional roles. For example, the conserved arginine of the bacterial and archaeal MDDs is replaced by glutamine in C. aerophile, threonine in A. thermophila and H. aurantiacus, and glutamate in Roseiflexus sp., R. castenholzii, Chloroflexus sp., C. aurantiacus, C. aggregans, O. trichoides, and H. volcanii (Figure 3). Interestingly, in the substrate ambiguous AthermMPD, the corresponding residue, Thr191, can interact through hydrogen bonding with the βphosphate group of (R)-MVAPP, though not with the single phosphate group of (R)-MVAP. In eukaryotes, the corresponding residue is likewise a conserved threonine (Thr217 in AthalMDD).

R71 R26 R193

K72 R78 AthermMPD AthalMDD SepMDD


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Figure 4. Positional coincidence of the side-chain guanidinium groups. Superposition of AthermMPD (pdb 6N0X, pink), AthalMDD (pdb 6N10, cyan), and Staphylococcus epidermidis MDD (pdb 4DPW, grey). ATP-�-S and MVAPP shown from Staphylococcus epidermis MDD structure. Atoms colored by atom type: oxygen, red; nitrogen, blue; phosphorus, orange; sulfur, yellow. Further, from multiple sequence alignments of the MDDs and MPDs, the conserved arginine of eukaryotic MDDs aligns with an arginine residue that occurs in all chloroflexi MPDs (Arg71 in AthermMPD) (Figure 3). However, structural superposition of AthalMDD and AthermMPD (Figure 4) clearly demonstrates that these arginines are not spatially or functionally equivalent. In AthalMDD, Arg78 is located in the first turn of α-helix 1 with its side chain extending toward the substrate-binding pocket (Figure 4). In contrast, in AthermMPD, Arg71 is located within a comparatively longer α-helix with its side chain extending toward the ATP binding pocket. The corresponding residue in bacterial and archaeal MDDs is likewise an arginine or conservatively replaced by a lysine, with the exception of Sulfolobus solfataricus MDD with a tyrosine at this position (Figure 3).28 The structure of ATP-complexed MDD from Staphylococcus epidermidis confirms this residue (Lys72) is involved in ATP binding, forming hydrogen bonds with the ribose 2’-hydroxyl and the heterocyclic oxygen of the ribose ring. We verified that Arg71 in AthermMPD likewise has a predominant role in ATP binding, as replacement by alanine caused a drastic increase in the KMATP (estimated to be >20 mM because a saturating concentration of ATP could not be attained) but had little impact on the KM of (R)-MVAP or (R)-MVAPP (Table 2). Engineering of HaMPD for (R)-MVAPP decarboxylase activity. We further explored the impact of targeted amino-acid replacements at important active-site residues in the highly specific HaMPD. The loss of the key histidine in the HaMPD H204M variant caused a 50-fold increase in KMMVAP to 1780 ± 250 μM but conferred no detectable gain in activity with (R)-


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MVAPP (Supplementary Table 1). This result is consistent with the effects of the corresponding H194M substitution in AthermMPD. However, the introduction of the key lysine in the Q24K HaMPD mutant elicited a modest level of activity with (R)-MVAPP, albeit with a high KMMVAPP of 6800 ± 1700 μM. The Q24K substitution in HaMPD also diminished the activity with (R)-MVAP, but to a substantially lesser degree than the corresponding N20K substitution in AthermMPD. Compared to each of the single mutants, the double mutant Q24K/H204M of HaMPD showed further increase in activity with (R)-MVAPP with a KM = 900 ± 300 μM, but with a surprisingly low kcat. Next, we introduced the arginine that was demonstrated to contribute to the substrate ambiguity of AthermMPD, and found that the HaMPD T30R single-site variant showed detectable turnover with (R)-MVAPP with a KM = 2300 ± 300 μM. Unfortunately, the triple mutant Q24K/H204M/T30R of HaMPD was unstable and could not be assayed. Functional analysis of altMVA1-pathway incorporation in Saccharomyces cerevisiae. The lack of genetic tools for organisms utilizing the altMVA pathway has limited studies of this pathway to in vitro recombinant enzyme assays. As an initial step in addressing this shortcoming, we tested whether components of the Anaerolinea thermophila altMVA1 pathway can functionally replace the correspondent components of the MVA pathway, employing S. cerevisiae as an in vivo model. S. cerevisiae uses the MVA pathway exclusively for IPP biosynthesis and accordingly this pathway is essential for viability. Deficiency in either of the two enzyme activities characteristic of the classical MVA pathway, PMK (encoded by ERG8) and MDD (encoded by ERG19), are deleterious for yeast growth. The essentiality of the MVA pathway in yeast provides a simple phenotypic read-out for assessing functional complementation by genes unique to the altMVA1 pathway.32


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For these experiments, two S. cerevisiae host deletion-strains, erg19-Δ::nat/p CEN URA3 ERG19 and erg8-Δ::kan erg19-Δ::nat/p CEN URA3 ERG8 ERG19, were constructed as described in the Methods section. For complementation analyses, the heterologous gene of interest, A. thermophila mpd or ipk, and for the comparison also Arabidopsis thaliana mdd1 or ipk, was placed in a low-copy number CEN plasmids, under the control of relevant yeast promoters (500 bp of the ERG8 promoter driving expression of ipk, and 500 bp of the ERG19 promoter driving expression of mpd or mdd). Following transformation into the appropriate yeast deletion-strain, we assessed whether the targeted component(s) of the yeast MVA pathway became dispensable for viability upon expression of a potentially complementary gene(s) from the altMVA1 (or Arabidopsis thaliana classical MVA) pathway. We first examined complementation of the yeast MDD-deletion. Plasmids expressing Athalmdd1 or Athermmpd were separately transformed into the erg19-Δ::nat/p CEN URA3 ERG19 strain and plated on medium containing the counter-selective drug 5-FOA to select for loss of the ERG19 URA3 plasmid. Both Athalmdd1 and Athermmpd rescue the lethality of the erg19-Δ deletion, even when expressed at normal (i.e. physiological) levels (Figure 5A). These results are consistent with in vitro activity assays demonstrating both AthalMDD1 and AthermMPD catalyze the decarboxylation of (R)-MVAPP to IPP with a catalytic efficiency similar to that previously reported for Saccharomyces cerevisiae MDD (ScMDD). The kcat/KMMVAPP for AthalMDD1, AthermMPD, and ScMDD are 1.2 × 105 s-1M-1, 1.6 × 104 s-1M-1, and 4 × 104 s-1M-1 respectively.

18,26

The result with Athalmdd1 reported here also

substantiates under more indigenous conditions a previous demonstration of the complementation of a Saccharomyces cerevisiae erg19-mutant by Athalmdd1, in which the strong promoter, PMA1, and a high-copy number plasmid were used.33


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A ERG19 erg19-Δ erg19-Δ + Athalmdd1 erg19-Δ + Athermmpd erg19-Δ + Athermmpd N20K/H194M erg19-Δ + Athermmpd R26V

B ERG8 ERG19 erg8-Δ erg19-Δ erg8-Δ erg19-Δ + Athalipk + Athalmdd1 erg8-Δ erg19-Δ + Athermipk + Athermmpd erg8-Δ erg19-Δ + Athermipk + Athermmpd N20K/H194M erg8-Δ erg19-Δ + Athermipk + Athermmpd R26V

Figure 5. Viability of Saccharomyces cerevisiae strains (A) erg19Δ::nat and (B) erg8Δ::kan erg19Δ::nat on 5-FOA expressing noted genes of interest. Serial dilutions of strains cultured overnight in leu drop out media were plated on complete minimal 5-FOA and photographed after 4 days of incubation at 30 oC. We next sought to restore viability to a yeast strain lacking the two distinguishing enzyme activities of the classical MVA pathway, PMK and MDD. Various heterologous ipk and mpd or mdd pairs were transformed into the erg8-Δ::kan erg19-Δ::nat/p CEN URA3 ERG8 ERG19 yeast deletion-strain. Viability was recovered in a strain expressing Athermipk and Athermmpd (Figure 5B). This result shows that in the absence of pmk and mdd of the yeast MVA pathway, native levels of expression of Athermmpd and Athermipk are sufficient to produce IPP for downstream biosynthetic needs of the yeast cell, including production of the essential membrane sterol, ergosterol. Furthermore, we have demonstrated for the first time that the set of enzyme activities representative of the complete altMVA1 pathway can serve as a functional substitute for the traditional MVA pathway. Notably, expression of Athalipk


25

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and Athalmdd1 in the erg8-Δ erg19-Δ yeast strain could not restore viability, indicating the Arabidopsis thaliana MDD from the MVA pathway in combination with an IPK cannot constitute a functional altMVA1-like pathway in vivo. We observed the same result for Athalmdd1 paired with Athermipk. These results are consistent with the poor activity of AthalMDD with (R)-MVAP and suggest that IPK must play an alternative role in isoprenoid biosynthesis in Arabidopsis.17,18 Lastly, we evaluated in vivo two AthermMPD variants demonstrating near exclusive specificity for (R)-MVAP (AthermMPD R26V) or (R)-MVAPP (AthermMPD N20K/H194M). We found that AthermMPD N20K/H194M but not AthermMPD R26V complemented erg19Δ::nat (Figure 5A). Notably, AthermMPD N20K/H194M required the activity of the native yeast PMK to provide a source of (R)-MVAPP, as this variant with Athermipk failed to restore viability to erg8-Δ::kan erg19-Δ::nat (Figure 5B). On the other hand, AthermMPD R26V together with AthermIPK complemented erg8-Δ::kan erg19-Δ::nat (Figure 5B). These results show the in vitro substrate specificities of the structure guided variants of AthermMPD are reflected in the conferred in vivo phenotypes, and emphasize the importance of these residues in defining substrate preference in MPDs and MDDs. Furthermore, the complementation of the MVA pathway by the altMVA1 pathway is not dependent of the substrate ambiguity of AthermMPD, but rather is dependent solely on the MPD activity of AthermMPD, in conjunction with IPK activity. Functional annotation of MDD/MPDs and incidence of the classical/altMVA pathways. Applying our new knowledge concerning the substrate specificity determinants of the (R)MVAP(P) decarboxylases, we extensively analyzed the available MDD-homolog sequences across the three domains of life to predict likely MPD or MDD function, and, thereby more


26


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definitively assess the incidence of the MVA and altMVA pathways. More specifically, we focused on the occurrence of the key active-site histidine and lysine residues, corresponding to AthermMPD His194 and AthalMDD Lys26. MDD-homolog sequences were identified by protein BLAST using as the search query a known MDD or MPD protein sequence from the domain being investigated. Misannotated MDD sequences, which bear the MPD-defining histidine residue and lack the (R)-MVAPP-binding lysine residue, were identified in Chloroflexi bacteria with MDD-homolog sequences as well as the archaeal classes, Halobacteria and Thermoplasmata. Notably, Chloroflexi, Thermoplasmata, and Halobacteria do not possess a PMK but instead encode an IPK, further hallmarks of an operative altMVA pathway. In Thermoplasmata, two distantly related homologs of MDD that share low aminoacid sequence identity (approximately 20%) with characterized MDDs and MPDs were identified. The recent enzymatic characterization of these MDD homologs from Thermoplasma acidophilum as mevalonate 3-kinase (M3K) and mevalonate 3,5diphosphate decarboxylase (MBD) delineate yet another alternative MVA pathway (altMVA2) (Figure 1). 11,34 Although Thermoplasma acidophilum (TaM3K) produces an atypical intermediate of the MVA pathway, mevalonate-3-phosphate, structural comparisons of AthermMPD, AthalMDD, and TaM3K (pdb 4RKS),35 demonstrate that TaM3K retains several active-site residues characteristic of the MPD enzymes, including AthermMPD His194, and also lacks the key active-site lysine of the MDDs (Figure 3, Supplementary Figure 5). A fundamental, distinguishing feature of TaM3K is a glutamate residue, Glu140, which replaces a conserved glycine residue occurring in both the MPDs and MDDs. The side chain of this glutamate projects into space corresponding to the


27

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binding site of the 5-phosphate(s) of the (R)-MVAP or (R)-MVAPP substrates of MPD and MDD, and thereby sterically excludes these phosphorylated substrates (Supplementary Figure 5). Indeed, the Glu140 carboxylate-group in TaM3K forms a network of interactions with the active-site histidine and arginine that mirrors the network formed by the 5phosphate(s) of (R)-MVAP(P) with the equivalent residues in AthermMPD (Supplementary Figure 5). Likewise, T. acidophilum MBD retains the key active-site histidine of MPD while the active-site lysine residue of MDDs is a tyrosine (Figure 3).

12,13

The observed active site

similarity of MBD and MPD is not surprising given that the substrate of MBD, mevalonate 3,5-bisphosphate, is an intermediate of the reaction catalyzed by MPD, and the two enzymes produce the same product, IP. A distinguishing feature of MBD is the lack of key ATP binding residues, because the MBD substrate bears a 3-phosphate and therefore the decarboxylation reaction catalyzed by MBD does not involve an ATP-dependent phosphorylation step.13 Lastly, the MVA pathway of the bacteroidetes Flavobacterium johnsoniae was recently characterized and found to include distantly related MVK and PMK homologs and a (R)-MVAPP specific MDD.3 Surprisingly, Flavobacterium johnsoniae MDD possesses not only the key lysine (Lys37) characteristic of MDDs, but also the histidine (His233) characteristic of MPDs. Furthermore, the lysine and histidine residues are highly conserved in bactoroidetes, occurring in 282 of 285 MDD homologs. Our kinetic data suggests the histidine residue is critical for (R)-MVAP recognition and missing in all previously characterized MDDs. In AthermMPD, the variant H194M resulted in a 25-fold increase in the KM of (R)-MVAP and a 5-fold increase in the KM of (R)-MVAPP compared to wild type (Table 2) suggesting the histidine though less critical for recognition of (R)-MVAPP than


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(R)-MVAP may contextually aid recognition of (R)-MVAPP. Our results establish that the distribution of the altMVA pathways in life is relatively narrow, whereas the majority of organisms unequivocally possess a complete, classical MVA pathway. The altMVA1 pathway appears to be limited to some but not all extremophiles (for example, the MVA pathway was shown to exist in the Archaea genus Sulfolobus4). The altMVA1 pathway is found in some thermophilic bacteria of the phylum Chloroflexi and the halophilic archaea Halobacteria. The altMVA2 pathway is present in the acidophilic archaea Thermoplasmata. the most prevalent of the altMVA pathways, AltMVA3, lacks a MDD/MPD homolog entirely, and is widely distributed amongst Archaea.14 Nevertheless, several important issues relating to IPP biosynthesis remain unresolved. In particular, a large number of organisms, including those of the green plant lineage and the archaeal genera Sulfolobus and Metallosphaera, enigmatically encode a homolog of IPK but possess homologs of MDD that are inconsistent with the MPD sequencesignature. The selectivity of Sulfolobus solfataricus MDDs for (R)-MVAPP has been previously verified.4 Furthermore, we confirmed that the two MDD homologs from Arabidopsis thaliana are highly specific for (R)-MVAPP.17 Based on our current analyses, MPD activity is unlikely to occur in plants. A further curiosity is the significant number of archaeal species that lack a homolog of both MDD and MPD despite the presence of many of the MVA pathway enzymes and also the altMVA pathway enzyme IPK. Recently, an additional altMVA pathway (altMVA3, Figure 1) was demonstrated in vivo for Aeropyrum pernix, in which IP production is dependent not on an MDD/MPD activity but instead on the activities of a MVAP dehydratase and UbiD-type decarboxylase.14 The identification of three alternative MVA pathways


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further highlights the complexity of IPP biosynthesis and the interesting evolutionary malleability of the penultimate and ultimate steps of the MVA pathways. CONCLUSION Mevalonate phosphate decarboxylase represents a distinct subclass of the GHMP kinase superfamily with an important role in the essential biosynthesis of IPP via one alternative MVA pathway. MPD enzymes are catalytically and evolutionarily very similar to MDD enzymes, and, have therefore been misclassified as possessing MDD activity in annotated databases. We demonstrate an annotated MDD from the Chloroflexi Anaerolinea thermophila possesses nearly equal catalytic efficiency for (R)-MVAP and (R)-MVAPP substrates. We present the first crystal structure of an enzyme with MPD activity, and through structural, sequence, and kinetic analyses, identify active site residues important for substrate discrimination. Comprehensive analyses of MDD/MPD homologs delineate the distribution of the two enzyme classes across the three domains of life. MPDs are predicted to exist in the archaea Halobacteria class in addition to the bacterial Chloroflexi phylum. On the other hand, in plants, annotated MDDs do in fact correspond closely to true functional MDDs, and thus, the absence of MPD activity indicates that altMVA1 pathway is not operative in the green plant lineage despite the universal occurrence of plant IPKs. METHODS Expression and Protein Purification. purified as reported previously.17

Arabidopsis thaliana mdd was expressed and

Anaerolinea

thermophila mpd, mvk, and ipk and

Herpetosiphon aurantiacus mpd were codon optimized and synthesized for expression in E. coli by Genscript. The genes were cloned into a modified pET28b plasmid (Novagen) encoding for a N terminal His8 tag by In-Fusion HD Cloning System(Takara). Mutations were


30


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introduced following the Q5 site-directed mutagenesis procedure (New England Biolabs). Expression constructs were transformed into BL21(DE3) E. coli (BioPioneer) and expressed in TB media (Millipore) supplemented with 50 g/mL kanamycin (BioPioneer) at 37˚C. When the OD600 reached 1.0 the temperature was lowered to 18˚C and expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (BioPioneer). After 15 - 20 h cells were collected by centrifugation and lysed by sonication in 50 mM Tris-HCl pH 8.0 buffer supplemented with 0.5 M NaCl, 20 mM imidazole, and 10% glycerol. Cellular debris was removed by centrifugation at 18,000 rpm for 1 h at 4˚C. Supernatant was purified with Ni-NTA His Bind resin (ThermoFisher Scientific) and bound protein was eluted with 50 mM Tris-HCl pH 8.0 buffer supplemented with 0.5 M NaCl and 250 mM imidazole. Eluted protein was buffer exchanged by dialysis into 50 mM Tris-HCl pH 8.0 and 0.2 M NaCl. The N-terminal His8 tag was removed with thrombin and remaining histidine tagged protein was removed by passing over Ni-NTA His Bind resin. Further purification by size exclusion chromatography with a Superdex 200 16/60 column (GE Lifesciences) developed in 50 mM Tris-HCl pH 8.0 supplemented with 0.2 M NaCl, and 2 mM DTT. Proteins were used immediately or flash frozen in liquid N2 and stored at -80˚C. Enzyme Assays. Decarboxylase and kinase activities of A. thaliana MDD, A. thermophila MPD, MVK, and IPK and H. aurantiacus MPD were analyzed by the lactate dehydrogenasepyruvate kinase coupled assay as detailed previously. 7,21 Reactions include 7 U of pyruvate kinase (Sigma-Aldrich), 10 U of lactate dehydrogenase (Sigma-Aldrich), 4 mM ATP (SigmaAldrich), 2 mM phosphoenolpyruvate (Sigma-Aldrich), 0.16 mM NADH (Roche), 8 mM MgCl2, and 100 mM KCl in 100 mM Na+-HEPES buffer pH 7.5 at varied concentrations of (R)-MVAP (Sigma-Aldrich) or (R)-MVAPP (Sigma-Aldrich). Reactions where the apparent KM of ATP


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was determined were conducted at 0.5 mM or 1mM of (R)-MVAP or (R)-MVAPP. Reactions were initiated by the addition of enzyme and monitored at 340 nm at 30˚C. Initial velocity data were fit using Prism software (GraphPad Software Inc.) to the Michaelis-Menten equation to determine KM and kcat. Product production was verified by reverse-phase chromatography on a Shimadzu UPLC-SQD-MS with a Supelco Ascentis express C8 15cm x 4.6 mm column (Sigma-Aldrich). Assays with 1 mM (R)-MVAP or (R)-MVAPP, 2 mM ATP, and 1 M enzyme in 100 mM Na+HEPES buffer pH 7.5 were quenched after 1 h by vortexing with chloroform. The aqueous layer was separated at 35 ˚C with 5 mM dihexylammonium acetate in water (A) and acetonitrile (B) as the mobile phases over a ten-minute gradient at 1 mL/min flow rate starting at 30% B. After 2 minutes at 30% B, mobile phase B was increased to 60% B over 5 minutes and then held constant at 60% B for 1 minute. Mobile phase B was then lowered to 30% to re-equilibrate the column before the next injection. Ions were detected in negative ion mode. Crystallization and Structure Solution. Crystallization trials with a set of screening conditions developed in-house were conducted by the hanging drop method. Typically, 1 L of protein at 10 mg/mL in 50 mM Tris-HCl pH 8.0, 0.2M NaCl, and 2 mM DTT was mixed with 1 L of each reservoir solution and incubated at 4˚C over 500 L reservoir solution. Diffraction-quality AthermMPD crystals were obtained after microseeding from a mixture of the protein with 0.1 M acetic acid pH 4.5, 3-5% PEG 20,000, 0.3 M ammonium acetate, and 5 mM of (R)-MVAP or (R)-MVAPP. AthalMDD crystals were obtained from a mixture of protein with 0.1 M sodium citrate pH 5.6, 0.2 M potassium sodium tartrate, and 2.0 M ammonium sulfate. Substrate bound structures were obtained from crystals cocrystallized with 5 mM


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(R)-MVAPP. Crystals formed after two days and were flash frozen in cryoprotectant of 17% ethylene glycol and reservoir buff plus substrate. X-ray diffraction data were collected at beamlines 8.2.1 and 8.2.2 of the Advanced Light Source at Lawrence Berkeley National Laboratory. Diffraction images were indexed and integrated with iMosflm 7.1.1,36 and the measured reflection intensities were scaled and merged using CCP4 Aimless.37 The initial structural elucidation of AthermMPD complexed with (R)-MVAP was obtained by molecular replacement with CCP4 MolRep using a search model derived from Staphylococcus aureus MDD (pdb entry 2HK2) with nonconserved amino-acid residues pruned using CPP4 chainsaw. Similarly, Arabidopsis thaliana MDD was obtained by molecular replacement using a search model derived from human MDD (pdb entry 3D4J). The structural models were refined with Phenix Refine and inspected against electron-density maps (of the SigmaA weighted 2mFo-DFc or mFo-DFc difference type, where m is the figure of merit and D is the Sigma-A weighting factor) and adjusted manually in Coot. Autobuilding was performed with CCP4 ARP/wARP.37 Subsequent structure determinations of other forms of AthermMPD, which were crystallized isomorphously, were initiated with the refined AthermMPD/(R)-MVAP model, after omission of ligands, water molecules, and the side chains of residues of specific interest. X-ray diffraction data processing and refinement statistics are displayed in Table 3.


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Table 3. X-ray diffraction data processing and refinement statistics AthermMPD (R)-MVAP complex

space group cell dimensions a, b, c (Å) α, β, γ (º) resolution (Å) I/σI CC1/2 Rmerge Rmeas Measure reflections Unique reflections completeness multiplicity Rwork/Rfree Average B-factors rms deviations bond lengths (Å) bond angles (deg) Ramachandran (%) Favored Outliers PDB ID

AthermMPD (R)-MVAPP complex Data collection P1 21 1

AthermMPD N20K/H194M (R)MVAPP complex

AthalMDD (R)-MVAPP complex

P1 21 1

P 41 21 2

80.80, 138.09, 105.13 90, 96.12, 90 46.03– 1.95 8.5 (1.6) 0.989 (0.547) 0.120 (0.624) 0.139 (0.799) 403730 (10659) 131416 (5384) 79.0 (65.7) 3.1 (2.0)

106.63, 106.63, 166.23 90, 90, 90 53.32 – 2.30 13.1 (1.4) 0.998 (0.615) 0.124 (0.909) 0.128 (1.052) 450428 (10276) 42293 (2744) 97.8 (96.0) 10.7 (3.7)

0.1722/0.1961 25.48

80.48, 137.5, 106.5 90, 97.67, 90 52.77 – 2.20 11.4 (1.7) 0.994 (0.617) 0.102 (0.516) 0.114 (0.679) 474291 (5319) 103991 (3426) 89.6 (59.9) 4.6 (1.6) Refinement 0.1724/0.2118 33.22

0.1674/0.2023 24.58

0.1897/0.2254 66.73

0.011 1.173

0.009 1.089

0.007 0.865

0.014 1.457

98.68 0.11 6N0X

97.76 0.21 6N0Y

97.42 0.21 6N0Z

94.24 0.50 6N10

P1 21 1 79.60, 135.63, 105.70 90, 97.72, 90 37.566 - 1.440 10.3 (1.1) 0.994 (0.291) 0.097 (0.899) 0.108 (1.271) 1377792 (13347) 361184 (11325) 90.4 (57.2) 3.8 (1.2)

a: Values in parentheses are for the highest-resolution shell. Genetic methods in S. cerevisiae. The chromosomal copies of ERG8/pmk and ERG19/mdd in a wild type S. cerevisiae strain, were replaced by kanamycin and nourseothricin resistance cassettes respectively to generate the erg19-Δ::nat and erg8-Δ::kan erg19-Δ::nat strains. Chromosomal deletions were confirmed by PCR and DNA sequencing, and viability was maintained by expression of ERG8 and ERG19 on an URA3 CEN plasmid. Genes of interest, including Arabidopsis thaliana mdd and Anaerolinea thermophila mpd and ipk were cloned into a LEU2 CEN plasmid with 500 bp of the ERG8 (for ipk) and ERG19 (for mpd or mdd) promoters and transformed into the appropriate deletion strain. Transformants were grown to saturation in –Leu media and a five-fold dilution series was plated on –Leu plates


34


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containing the counter-selective drug 5-FOA to select for loss of the URA3 plasmid, to assess viability of yeast strains expressing ipk, mpd or mdd in the absence of the yeast MVA pathway . Plates were analyzed after four days of incubation at 30 oC. Bioinformatic Analysis. Sequence alignment was generated with T-Coffee using default settings.38 Sequences were obtained from Uniprot or National Center for Biotechnology Information. BLAST was used to identify MPD and MDD sequences. Accession Codes. Atomic coordinates and structure factors for AthermMPD (R)-MVAP, AthermMPD (R)-MVAPP, AthermMPD N20K/H194M (R)-MVAPP, and AthalMDD (R)MVAPP complexes have been deposited in the Protein Data Bank with accession codes 6N0X, 6N0Y, 6N0Z, and 6N10 respectively. ABBREVIATIONS. altMVA, alternative mevalonate; AthalMDD, Arabidopsis thalians mevalonate

5-diphosphate

decarboxylase;

AthermMPD,

Anaerolinea

thermophila

mevalonate 5-phosphate decarboxylase; HaMPD, Herpetosiphon aurantiacus mevalonate phosphate decarboxylase; GHMP, galactokinase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase; IPK, isopentenyl phosphate kinase; IP, isopentenyl phosphate; IPP, isopentenyl diphosphate; MBD, mevalonate 3,5-diphosphate decarboxylase; MDD, mevalonate diphosphate decarboxylase; M3K, mevalonate 3-kinase; MEP, methylerythritol phosphate; MPD, mevalonate phosphate decarboxylase; (R)-MVA, (R)-mevalonate; (R)MVAP, (R)-mevalonate 5-phosphate; (R)-MVAPP, (R)-mevalonate 5-diphosphate; MVK, mevalonate kinase; pdb, protein data bank; PMK, phosphomevalonate kinase. AKNOWLEDGMENTS. This work was funded by the Howard Hughes Medical Institute (S.T.T, G.V.L. and J.P.N.), United States National Science Foundation grant EEC-0813570 (J.P.N.), the Salk Institute Arthur and Julie Woodrow Chair (J.P.N.), United States National Science


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Foundation Graduate Research Fellowship DGE-1650112 (J.W.L.), National Institutes of Health grants R01 AG-11728 (V.L.), and P30 CA-014195 (to the Salk Institute Cancer Center). The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DEAC02-05CH11231 Supporting Information Available: This material is available free of charge via the Internet. LCMS analysis of AthermMPD, HaMPD, and AthermMVK assays, full multiple sequence alignment of Figure 3, active site comparison of MPD, MDD, and M3K, and steady-state kinetic parameters for HaMPD mutants. REFERENCES (1) Gershenzon, J., and Dudareva, N. (2007) The function of terpene natural products in the natural world. Nat. Chem. Biol. 3, 408–414. (2) Lombard, J., and Moreira, D. (2011) Origins and early evolution of the mevalonate pathway of isoprenoid biosynthesis in the three domains of life. Mol. Biol. Evol. 28, 87–99. (3) Hayakawa, H., Sobue, F., Motoyama, K., Yoshimura, T., and Hemmi, H. (2017) Identification of enzymes involved in the mevalonate pathway of Flavobacterium johnsoniae. Biochem. Bioph. Res. Commun. 487, 702–708. (4) Nishimura, H., Azami, Y., Miyagawa, M., Hashimoto, C., Yoshimura, T., and Hemmi, H. (2013) Biochemical evidence supporting the presence of the classical mevalonate pathway in the thermoacidophilic archaeon Sulfolobus solfataricus. J. Biochem. 153, 415–420. (5) Boronat, A., and Rodriguez-Concepcion, M. (2015) Terpenoid biosynthesis in prokaryotes. Adv. Biochem. Eng./Biotechnol. 148, 3–18.


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Substrate Specificity and Engineering of Mevalonate 5-Phosphate

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