Bacterial Branched-Chain Amino Acid Biosynthesis: Structures

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Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms and Drugability Tathyana Mar Amorim Franco, and John S Blanchard Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00849 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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Biochemistry

Bacterial branched-chain amino acid biosynthesis: Structures, mechanisms and drugability

Tathyana M. Amorim Franco and John S. Blanchard*

Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10805, USA.

KEYWORDS: branched-chain amino acids, enzymes, mechanism, biosynthesis, bacteria, antibacterial, structure, function

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ABSTRACT The eight enzymes responsible for the biosynthesis of the three branched-chain amino acids Lisoleucine, L-leucine and L-valine were identified decades ago using classical genetic approaches based on amino acid auxotrophy. This review will highlight the recent progress in the determination of the three-dimensional structures of these enzymes, their chemical mechanisms and insights into their suitability as targets for the development of antibacterial agents. Given the enormous rise in bacterial drug resistance to every major class of antibacterial compound, there is a clear and present need for the identification of new antibacterial compounds with nonoverlapping targets to currently used antibacterials that target cell wall, protein, mRNA and DNA synthesis.


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INTRODUCTION

Bacteria can synthesize all twenty proteinogenic amino acids, including the nine essential amino acids required for mammalian growth. In general, enzymes involved in the biosynthesis of amino acids are essential for the growth and survival of bacteria. In M. tuberculosis, for example, deletions of genes involved in the synthesis of the essential branched-chain amino acid (BCAA) L-leucine generated a successful attenuated strain that was also protective of mice challenged with a virulent strain of the bacilli 1. High density mutagenesis and in vitro inhibitory studies have also found that the biosynthetic pathway of the three BCAAs; L-isoleucine, L-leucine and L-valine, is vital for the growth and survival of M. tuberculosis 2, 3. These data make the enzymes involved in the BCAA biosynthetic pathway in M. tuberculosis and other pathogenic bacteria of great relevance for prospective antibacterial drug development. Although BCAAs are structurally similar amino acids containing aliphatic side chains, their propensity to be found in protein structures are quite different. L-valine and L-isoleucine for example, are overrepresented in β-sheets, while L-leucine is found primarily in α-helices, loops, and leucine zippers 4. The small differences in size, hydrophobicity and degree and position of branching of the side chains, explains why these amino acids are not interchangeable in proteins. In fact, the substitution of one BCAA for another may in some cases lead to diseases such as alterations in the plasm lipid profile, hypocalciuria and cardiomyopathy 4. The biosynthetic pathway of BCAAs is a very efficient pathway when compared to pathways leading to the synthesis of other amino acids. While, other pathways require many enzymes to synthesize a single amino acid (e.g., nine enzymes are required for the conversion of L-aspartate to L-lysine), the BCAA biosynthetic pathway requires only eight enzymes for the synthesis of all three BCAAs (Figure 1). 3 ACS Paragon Plus Environment

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OH H2N

CO2

L-threonine ilvA

O

CO2

O

2-ketobutyrate pyruvate

ilvBN

O

CO2

HO

2(S)-aceto-2-hydroxybutyrate

CO2

HO

O

2-keto-3-methylvalerate

CO2 HO

CO2

ilvD

CO2

CO2

2-ketoisovalerate

H2N

CO2

L-valine

CO2

3-isopropylmalate leuA

leuB

O

CO2

2-ketoisocaproate ilvE

ilvE

ilvE

L-isoleucine

leuCD

2,3-dihydroxyisovalerate

ilvD

CO2

CO2 2-isopropylmalate

OH

2,3-dihydroxy-3-methylvalerate

H2N

CO2

ilvC

OH

O

OH CO2

AcCoA

2(S)-acetolactate

ilvC

HO

pantothenate

ilvBN

pyruvate

O HO

CO2

pyruvate

H2 N

CO2

L-leucine

Figure 1. The biosynthetic pathway of the branched-chain amino acids in bacteria and its feedback regulatory points.


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Four of the eight enzymes are shared for the synthesis of all three BCAAs (IlvB/N; IlvC, IlvD and IlvE), while three are only responsible for the synthesis of L-leucine (LeuA, LeuC/D and LeuB), and one is specifically involved in L-isoleucine biosynthesis (IlvA). In this review, each enzyme is discussed individually, focusing on structure and function as well as their potential as antibacterial drug targets (Table 1).

Enzyme L-threonine dehydratase/deaminase Acetolactate (acetohydroxyacid) synthase

Gene Name ilvA

ilvBN

PDB Rv Proven ID number Drugability 4.3.1.27 3WQE, Rv1559 ** 1TDJ, 4PB4 Rv3003c Herbicides 2.2.1.6/4/1.3.18 1YI1, 1OZH, (B1) , 1OZF Rv4370c (B2), Rv3002c (N) EC number

Keto acid isomeroreductase

ilvC

1.1.1.86

Dihydroxyacid dehydratase Isopropylmalate synthase

ilvD

4.2.1.9

leuA

2.3.3.13

Isopropylmalate isomerase

leuCD

4.2.1.33

Isopropylmalate dehydrogenase

leuB

1.1.1.85

Branched-chain

ilvE

2.6.1.42

4YPO, Rv3001c 1QMG, 1YRL, 1SR9 Rv0189c 3U6W, 3FIG, 4OV9 3Q3W, 3H5E, 3H5H, 3H5J and 2HCU (C); 3H5H (D) 3UDU, 1OSJ, 1W0D 1A3G,

Herbicides

**

Rv3710

**

Rv2988c (C), Rv2987c (D)

**

Rv2995c

**

Rv2210c

PLP 5

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3HT5, 5U3F

aminotransferase

inactivators

Table 1. Summary table. Description of the genes/enzymes involved in BCAA biosynthestic pathway. **inhibition studies and compounds have not been developed or tested.

1. IlvA-Threonine Dehydratase/Deaminase The first committed step in the biosynthesis of L-isoleucine is catalyzed by the ilvAencoded threonine dehydratase/deaminase (EC:4.3.1.19; TD). This enzyme plays a very important role in the biosynthetic pathway of BCAAs in microorganisms and plants. The pyridoxal 5’-phosphate dependent (PLP)-enzyme is responsible for the conversion of threonine (or serine) to 2-ketobutyrate (or pyruvate) and ammonia. TD was one of the first examples of metabolic control via negative feedback in microorganisms. This was due to studies performed in the 1950s

5, 6

, where it was established that the presence of the end-product, L-isoleucine, in the

growth media, reduced the activity of TD in Escherichia coli. This work led to the proposal that not only product or substrate analogues could inhibit an enzyme competitively, but that inhibition could be accomplished by a downstream product in a regulatory mechanism. The understanding of how such a structurally different molecule was capable of inhibiting TD in a competitive manner remained unclear for many years. The unusual non-Michaelis-Menten kinetics displayed by TD as a function of L-threonine concentration was noted early in the studies of this enzyme 6. Changeux reported completely different kinetic patterns in the presence of L-threonine and L-isoleucine which led him to propose the existence of two separate sites in the enzyme: one


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Biochemistry

site (site A) where L-threonine binds at the catalytic site and a second site (site B) for the binding of L-isoleucine 7. Changeux suggested that whenever L-threonine was bound to site A, the enzyme was active to perform its catalytic events. In addition, the affinity of the enzyme for Lthreonine would be increased whenever L-threonine was also bound to site B, in a cooperative way. On the other hand, if L-isoleucine was bound to site B, the affinity of the enzyme for Lthreonine at site A was decreased, and the enzymes’ activity diminished. These remarkable observations led to the initial proposal of allosteric regulation 8. Numerous studies have confirmed the sigmoidal initial velocity kinetics of the reaction as a function of L-threonine concentration

7, 9

. The sigmoidal nature of the kinetics is also altered in the presence of the end

products L-isoleucine and L-valine 9. While L-isoleucine acts as an allosteric inhibitor, L-valine allosterically activates the enzyme. Other studies have shown that TD is competitively inhibited by aminothiols

10

, including L-cysteine, which acts as an inactivator of the E. coli enzyme

11

.

TD’s inactivation by L-cysteine is restored upon supplementation with L-threonine, and bacterial growth is further improved by L-isoleucine supplementation. The chemical mechanism of TD starts with the attack of the α-amino group of L-threonine on the Schiff base in the active site, resulting in transimination and external aldimine formation. A proton abstraction from C-α, followed by water elimination leads to the formation of a product Schiff base. Reverse transimination of the external product aldimine by an enzyme lysine residue leads to the regeneration of the original internal aldimine and the release of an enamine. The tautomerization of the enamine forms 2-iminobutyrate which upon hydrolysis, releases ammonia and 2ketobutyrate (Scheme 1).


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Scheme 1. Chemical mechanism of threonine dehydratase/deaminase. Site-direct mutagenesis studies of the tetrameric enzyme consisting of 514-residue monomer chains

9

confirmed what was previously proposed with regard to its effector sites

12

.

Using these data, a more complex model to explain the homotropic cooperativity observed in TD was developed. The cooperativity profile is a consequence of the greater affinity of the substrates and analogues for the regulatory sites rather than for the catalytic sites

13

, suggesting that the

allosteric change observed from the low to the high activity state happens synchronously and progressively throughout the range of L-threonine concentrations. The 2.8 Å resolution crystal structure of TD from E. coli 14 revealed detailed implications for the allosteric mechanism. The 56 kDa enzyme belongs to the type II-fold of PLP-dependent enzymes, as a result of its sequence and structural similarities to the enzymes belonging to this family. The chemical reaction catalyzed by TD, β-elimination, is also a common feature shared among type II-fold PLP-dependent enzymes. TD is organized into two different domains: a larger N-terminal catalytic domain, which contains the PLP cofactor bound to Lys62 as a Schiff base 14; and, a smaller C-terminal regulatory domain. Each of the C-terminal regulatory domains in E. coli TD has nonequivalent effector-binding sites and the allosteric regulation is proposed to be Ile/Val concentration dependent 15 (Figure 2).


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Biochemistry

C-terminal allosteric domain

PLP Lys62

N-terminal catalytic domain

Figure 2. The 2.8 Å resolution three-dimensional structure of threonine dehydratase/deaminase (IlvA) from E. coli (PDB code 1TDJ). The structure is colored by secondary structure: β- sheets (light magenta), α-helices (teal), and loops (yellow). The catalytic (or site A as described by Changeux)7 and allosteric domains (or site B as described by Changeux )7 are labeled and a close-up view of the active site containing the PLP-cofactor bound as a Schiff base to Lys62 is shown.

The catalytic product of L-serine dehydration by TD, α-aminoacrylate, inhibits the common, final enzyme in the pathway, the PLP-dependent enzyme, branched-chain aminotransferase (BCAT) IlvE

16, 17

through a mechanism-based type of inhibition

17

. Studies have shown that

RidA (YjgF/YER057c/UK114) proteins can however, prevent this toxic enamine from building up in the cell and protect against BCAT inhibition 17, 18. In M. tuberculosis and Bacillus subtilis (B. subtilis) the essentiality of this enzyme has been demonstrated. The deletion of the ilvA gene in B. subtilis generates isoleucine auxotrophy 19 while the downregulation of the M. tuberculosis enzyme leads to growth impairment and increased susceptibility to stress. The ilvA knockdown strain of M. tuberculosis H37Ra is also


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more sensitive to antibiotic treatment, which may point to a synergistic potential for targeting this enzyme in combination with other therapeutic targets in the treatment of tuberculosis (TB).

2. IlvB/N- Acetolactate (acetohydroxyacid) Synthase The second enzyme in the biosynthesis of isoleucine is also the first universally shared enzyme in the biosynthetic pathway of BCAAs. The ilvBN-encoded acetohydroxyacid synthase (EC 2.2.1.6; AHAS), also known as acetolactate synthase, catalyzes the formation of 2acetolactate or 2-aceto-2-hydroxybutyrate from the decarboxylation of pyruvate and its condensation with either pyruvate (on the valine pathway branch) or 2-ketobutyrate (on the leucine pathway branch), respectively 20. AHAS requires three different cofactors to catalyze its reaction: thiamin diphosphate (ThDP), flavin adenine dinucleotide (FAD) and a magnesium ion (Mg2+). AHAS is present solely in autotrophic organisms, and in some such as E. coli and S. typhimurium, three different isozymes may be expressed: AHAS I (encoded by the ilvBN genes) 21-23

28

, AHAS II (encoded by the ilvGM genes) 24-27 and AHAS III (encoded by the ilvIH genes) 26,

. However, due to different chromosomal genetic mutations, AHAS II from E. coli

AHAS III from Salmonella typhimurium (S. typhimurium)

30

29

and

are inactive proteins. In the M.

tuberculosis genome, four catalytic AHAS subunits (ilvB1, Rv3003c; ilvB2, Rv3470c; ilvG, Rv1820; and ilvX, Rv3509c) and one regulatory subunit (ilvN, Rv3002c) have been annotated and the genes are dispersed along the chromosome 31, 32. A putative small regulatory subunit has also been also identified (ilvH) and is believed to be the regulatory subunit of ilvB2, based on similarities with the E. coli enzymes

33

. The ilvB1-ilvN pair displays the enzymatic

characteristics expected for the AHAS involved in the biosynthesis of BCAAs, including


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Biochemistry

cofactor requirements. On the other hand, ilvB2, ilvG and ilvX are implicated in a catabolic pathway leading to the production of 3-hydroxy-2-butanone and/or 2,3-butanediol 32 and will not be discussed here. AHAS belongs to the pyruvate oxidase (PO)-like subfamily, displaying structural similarities with enzymes of this class

34, 35

. Several crystal structures of AHAS have been

deposited to the Protein Data Bank (PDB). The enzyme is a heterodimer, comprised of two subunits, a large catalytic subunit (ilvB) of approximately 60-70 kDa, and a small regulatory subunit (ilvN) with its molecular weight estimated as 10-54 kDa 36. The large subunit of AHAS, ilvB, has activity by itself and binds ThDP in a V- conformation

36

that leads to a close contact

between the C2 of the thiazolium ring and the N4′ atom of the pyrimidine moiety. This is a common feature of ThDP-dependent enzymes. The active site of the yeast AHAS, which shares 42% sequence identity with M. tuberculosis AHAS, is located at the dimer interface. ThDP is oriented by hydrogen bonds and Van der Waals interactions, including the highly-conserved Lglutamate residue which is Glu85 in M. tuberculosis. This residue is implicated in catalysis as demonstrated by several mutagenesis studies 37-39. Glu85 substitution by alanine and by isosteric or isofunctional amino acid residues, e.g., L-glutamine and L-aspartate, respectively, led to a dramatic decrease in the activity of the enzyme in comparison to the wild-type

39

. Amino acid

substitutions of conserved residues located in the immediate proximity of Glu85, His84 and Gln86, also led to a decrease in AHAS activity, suggesting that these residues are involved in the stabilization of the Glu85 side chain, keeping it interacting with the N1’ atom of the ThDP Mutagenesis studies of Arg318 led to complete inactivation of the protein

40

39

.

while the

substitution of another highly conserved residue Pro126, demonstrates its importance for ThDP binding

36

. The FAD binding-site in AHAS is located in one monomer and seems not to be


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implicated in dimer stabilization 41, but is required for structural integrity 42. The adenine ring of FAD is solvent exposed while the isoalloxazine ring is buried adjacent to the active site

43-45

.

Several crystal structures of AHAS from Klebisiella pneumoniae have been deposited (PDB codes SDX6, SD6R, 1OZF, 1OZG, and 1OZH) from this pathogenic bacteria 41 (Figure 3).

Figure 3. The 2.3 Å resolution three-dimensional structure of acetolactate (acetohydroxyacid) synthase (IlvBN) from Klebisiella pneumoniae (PDB code 1OZF) 41. The structure is colored by monomer. A close-up view of the active site containing the ThDP- bound cofactor and the green sphere representing the Mg2+ ion. Steady-state kinetic studies of AHAS from M. tuberculosis and other eubacteria 46, reveal non-hyperbolic behavior. Saturation curves varying pyruvate revealed that the large catalytic subunit of AHAS (ilvB1) alone displays positive cooperativity (Hill coefficient = 2.0) while the heterodimeric holoenzyme displays negative cooperativity (Hill coefficient = 0.6)

47

. Titrations

of the small regulatory subunit (ilvN) increased the specific activity of the large catalytic subunit (ilvB1) to values corresponding to that observed for the holoenzyme

47

. The M. tuberculosis 12

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Biochemistry

AHAS, as well as its orthologues from other bacteria, is regulated by negative feedback by branched-chain amino acids. In the presence of L-valine and to a lesser extent, L-isoleucine, the holoenzyme is partially inhibited while L-leucine has no effect on the activity of the enzyme 47. The catalytic mechanism of AHAS starts with the binding of pyruvate and ionization of ThDP at the active site, followed by the addition of ThDP to C2 of pyruvate. The covalent tetrahedral intermediate, 2-lactyl-ThDP (LThDP) is decarboxylated in the following step. The decarboxylated product, hydroxyethylthiamine diphosphate (HEThDP), attacks the carbonyl of the second substrate, which can be either pyruvate or 2-ketobutyrate, to form the acetohydroxyacid-ThDP (AHAThDP) adduct. The final step involves release of ThDP from either product, acetolactate or acetohydroxybutyrate. FAD does not play a catalytic role in the mechanism of AHAS, however, during the catalytic process, it can undergo reduction as a consequence of an oxygen-dependent side reaction 48 (Scheme 2).

R2

R2

R2

O

+ R1 N

S

thiamin

+ O

CO2

pyruvate

R1 N

R1 N

S O OH

HO

CO2

H+

S

CO2 2-aceto-2-hydroxybutyrate

2-ketobutyrate

Scheme 2. Chemical mechanism of acetolactate (acetohydroxyacid) synthase.

AHAS is a very attractive target for drug development and inhibition, due to its absence in mammals and thus, reduced potential for toxicity 3. Since the serendipitous discovery of AHAS as the target of sulfonylurea (SU) herbicides in plants sulfonylureas as antibacterial agents have been tested

24

49

, the potency of these

. The M. tuberculosis AHAS is also

inhibited by sulfonylureas and other AHAS-specific inhibitors 3, however the inhibitory activity of these are inferior to that of the standard antibiotics used in the treatment of TB 47. However, 13 ACS Paragon Plus Environment

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monosubstituted sulfonylureas displayed potent inhibitory activity against clinical tuberculosis strains, including multidrug-resistant (MDR)- and extensively drug-resistant (XDR)-TB isolates 50

. The druggability of the M. tuberculosis AHAS has led to the screening and design of

compounds targeting the bacilli’s enzyme, including ssDNA aptamers

3, 40, 50-55

. Many of these

inhibitors are active against both the enzyme and resistant strains, with minimal inhibitory concentration (MIC) values similar to those of the standard antibiotics

40, 53

. Molecular docking

experiments revealed that most of these inhibitors likely bind outside of the active site

53

, in

agreement with what was previously reported for SU herbicides 40, 43, 47, 53.

3. IlvC- Keto Acid Isomeroreductase The second enzyme in the BCAA biosynthesis pathway is the ketol-acid isomeroreductase (EC 1.1.1.86; KARI). The enzyme converts the products of AHAS, 2acetolactate or 2-aceto-2-hydroxybutyrate, to their respective 2,3-dihydroxy products. The reaction catalyzed by KARI was first thought to be performed by two different enzymes, the first catalyzing the alkyl migration followed by keto acid reduction. However, in 1961 it was reported that purified KARI from S. typhimurium was the only enzyme involved in the conversion of either 2-acetolactate or 2-aceto-2-hydroxybutyrate to products

56

. The properties of the enzyme

were also investigated and shown to be similar to those reported for the E. coli 57, 58, Neurospora crassa

57

and Saccharomyces cerevisiae

59

enzymes. KARI from all sources absolutely required

Mg2+, used NADPH (reduced nicotinamide adenine dinucleotide phosphate) as a reductant and both NADP+ and 2,3-dihydroxyacid exhibited product inhibition

56

. The chemical

bifunctionality, isomerization and reduction, of KARI have been also investigated through sitedirect mutagenesis 60.


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Biochemistry

There are two different classes of KARI enzymes: class I enzymes are composed of ca. 340 amino acids while class II enzymes are larger at approximately 490 amino acid residues. The class II KARIs are present in some bacteria, including E. coli, and all plants 61. M. tuberculosis KARI belongs to the class I of enzymes

61

, as does Pseudomonas aeruginosa

62

, Spodoptera

exigua 63, and other bacteria 64, 65. In all cases, two different domains, N-terminal and C-terminal, are present. The N-terminal domain folds into a mixed β-sheet flanked on both sides by helices in a nucleotide-binding Rossmann fold. The C-terminal domain is composed of eight α-helices forming a knotted structure 61. The M. tuberculosis KARI (MtKARI) crystal structure was solved at 1.0 Å resolution and is a dimer in solution

61

(Figure 4). The active site of the MtKARI is

formed upon dimerization of the protein, a characteristic feature of the class I KARI enzymes 6165

. Two Mg2+ ions are present at the active site, and are separated by approximately 5 Å. The first

metal ion is coordinated by Asp188 and Glu192 along with four water molecules, while the second one is also coordinated by Asp188, Glu224 and Glu228, and three water molecules

61

.

The Mg2+ ions, as well as the active site of M. tuberculosis KARI, are solvent exposed and allow easy access for substrate binding. The NADPH binding site consists primarily of residues in the N-terminal Rossmann-fold, although some contacts from the C-terminal domain may influence NADPH binding 61. As opposed to the larger class II KARI enzymes, M. tuberculosis KARI does not undergo significant conformational changes upon NADPH binding 61.


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E224 (A) Rossmann fold D188 (B)

E228 (A)

E192 (B)

Figure 4. The 1.0 Å resolution three-dimensional structure of keto acid isomeroreductase from M. tuberculosis (PDB code 4YPO). The homodimer is colored by monomer and the loops are colored in yellow. The domain containing the Rossmann fold is highlighted. A close-up view of the active site is shown highlighting the main residues coordinating the Mg2+ ions (green spheres). The active site is at the juncture of the N-terminus of one monomer and the C-terminus of the other monomer (circle).

KARI enzymes exhibit high stereospecificity for the S isomers of their substrates Kinetic constants for the MtKARI enzyme have been determined 60,

reported for the E. coli enzyme

68,

69

61

66, 67

.

and compared with values

. KARI’s reaction can be monitored

spectrophotometrically by observing the oxidation of NADPH at 340 nm. As a general feature of both class I and class II KARI enzymes, the activity of KARI is higher with 2-aceto-2hydroxybutyrate than with 2-acetolactate E. coli and S. typhimurium

71

61, 69, 70

. At saturating concentrations of Mg2+, both the

enzymes follow an ordered kinetic mechanism where NADPH

binds first followed by 2-acetolactate or 2-aceto-2-hydroxybutyrate. The binding order of Mg2+ and NADPH however, is random

69

. The isomerization chemistry involves base-assisted

deprotonation of C2 hydroxyl, and methyl or ethyl group migration to C3 to generate the α-ketoβ-hydroxyacid. NADPH transfers the pro-S hydrogen as a hydride ion to reduce the keto acid to 16 ACS Paragon Plus Environment

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Biochemistry

the C2 hydroxyl product

61, 69

. Solvent kinetic isotope effect data suggest that reduction of the

intermediate is not the rate-limiting step, but more likely the isomerization/alkyl migration step preceding the reduction reaction

69

(Scheme 3). In the presence of 2-acetolactate, E. coli KARI

specifically requires Mg2+ ion for activity, however, Mn2+ can substitute for Mg2+ when the substrate utilized is 2-aceto-2-hydroxybutyrate 69. There are no data concerning the activity of M. tuberculosis KARI with different divalent metal ions.

Scheme 3. The chemical mechanism of keto acid isomeroreductase.

The potential of KARI as an antibacterial drug target has been established. Some herbicidal

compounds,

such

as

the

KARI

transition

oxalylhydroxamate (IpOHA), also display antibacterial activity

state 72

analogue

N-isopropyl

and is a very potent inhibitor

of the E.coli KARI. The intermediate analogue binds to the active site of the enzyme

40, 43, 47, 53

and inactivates the enzyme in a time-dependent fashion, leading to the formation of an irreversible enzyme-inhibitor complex 68. MtKARI is also inhibited by the tight binding inhibitor IpOHA and displays a KI of approximately 98 nM 61, validating the targetability of this enzyme in M. tuberculosis. The analogue also displayed good inhibitory activity against clinical drugresistant strains of M. tuberculosis, however, its effect was not superior to the current drugs used to treat the disease 3. Several compounds have been designed for herbicidal purposes

73-76

and


Biochemistry

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Page 18 of 51

could also be explored in the inhibition of KARI from pathogenic bacteria, including M. tuberculosis.

4. IlvD- Dihydroxyacid Dehydratase Preceding the final transamination step that leads to the synthesis of L-isoleucine and Lvaline, and the fifth of the seven steps leading to the production of L-leucine, the ilvD-encoded dihydroxy acid dehydratase (EC 4.2.1.9; DHAD) is the enzyme responsible for the synthesis of 2-keto-3-methylvalerate and 2-ketoisovalerate. DHAD is also required for the synthesis of pantothenate, since one of its products, 2-ketoisovalerate, is a precursor to this pathway. The importance of DHAD was first investigated by using a partially purified enzyme from E. coli extracts 77, where the first evidence for the requirement of a ferrous ion or other divalent cation was identified 77. DHAD has been studied in several organisms including bacteria plants

80, 85-88

59, 78-83

, fungi

59, 84

. The most well studied DHAD from a bacterial source is the E. coli enzyme

Stereospecificity studies of the S. typhimurium DHAD transcriptional studies of the B. subtilis DHAD

92

79

and

89-91

.

, as well as transcriptional and post-

have also been performed. S. typhimurium

DHAD demonstrates absolute stereospecificity since only 2R-keto-3R-methylvalerate and 2Rketoisovalerate support bacterial growth 79. DHADs are homodimeric iron-sulfur cluster enzymes with monomer molecular weights ranging from 60-70 kDa 83, 90. Although plants 88 have been shown to contain a [2Fe-2S] cluster, bacterial DHADs contain a [4Fe-4S] cluster, but in both cases, the iron-sulfur cluster is absolutely required for catalysis

83, 88, 90, 91

. Enzymes having iron-sulfur clusters as cofactors are

highly oxygen sensitive, and the oxidative disruption of the cluster in bacterial DHAD leads to


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Biochemistry

the complete inactivation of the protein

89, 90

. The activity of E. coli DHAD under aerobic

conditions decreased by 100% in two hours 90. When the effects of nitric oxide (NO) were tested on the E. coli DHAD, the rapid reaction of NO with the iron-sulfur cluster of the enzyme was synchronous with the formation of a DHAD-dinitrosyl-iron complex which completely inactivates the enzyme even under anaerobic conditions. The rate of NO reaction with the ironsulfur cluster of DHAD is faster than the rate of its oxidation by oxygen, and the addition of glutathione to the reaction, carried out under anaerobic or aerobic conditions, does not prevent the enzyme from being inactivated by NO

93

. The sensitivity of DHAD enzymes to oxygen is

likely the reason why, to date, no three-dimensional crystal structures have been reported from any organism. The catalytic mechanisms of both S. typhimurium and E. coli DHADs have been reported and in both cases catalysis is dependent of the [4Fe-4S] cluster. The cluster acts as a Lewis acid and the C3-hydroxyl group of the 2,3-dihydroxy-valerate substrates binds as a ligand to the cluster, activating it for β-elimination of a water molecule upon C2 proton abstraction. This reaction results in the formation of an enol intermediate that tautomerizes with stereospecific C3 protonation

to

generate

the

keto

acid

product

79

.

The

stereospecificity

of

the

tautomerization/protonation strongly suggests that this step occurs in the proteins’ active site prior to product release (Scheme 4) 90, 94.


Biochemistry

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Page 20 of 51

Scheme 4. The chemical mechanism of the [4Fe-4S] cluster protein dihydroxyacid dehydratase. M. tuberculosis DHAD (MtDHAD) is a 118 kDa homodimer in solution and like the E. coli enzyme contains a bacterial type [4Fe-4S] cluster. The enzyme is also oxygen and NO sensitive, and the growth of M. tuberculosis treated with NO can only be restored upon supplementation with the three BCAAs

83

. Downregulation of the ilvD gene in M. tuberculosis

generated a partially auxotrophic strain, unable to grow in mice but still capable of persisting in the tissue

83

. The downregulation of the gene allows for the suboptimal synthesis of branched-

chain amino acids, and therefore a knockout study could clarify the potential of the ilvD gene for auxotrophy. The essentiality of this enzyme for growth and survival of mycobacteria makes this enzyme a very interesting potential antibacterial target, since its inhibition impairs not only the synthesis of BCAAs but also pantothenate and consequently the synthesis of CoA, for which pantothenate is a precursor.

5. LeuA- Isopropylmalate Synthase The first enzyme in the branch that leads to L-leucine biosynthesis is the leuA-encoded isopropylmalate synthase (EC 2.3.3.13, IPMS). IPMS catalyzes the first committed step in the synthesis of L-leucine with the conversion of 2-ketoisovalerate and acetyl-CoA to 2isopropylmalate and CoA. The enzyme was first purified from S. typhymurium in 1969 and some


Page 21 of 51

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Biochemistry

kinetic properties of this enzyme were characterized

95

, such as the optimum pH of the enzyme

(pH 8.5) and evidence for an allosteric mechanism. IPMS has been extensively studied in M. tuberculosis (MtIPMS). MtIPMS was crystalized and the structure was solved at 2.0 Å resolution (Figure 5)

96

. The homodimer is

composed of two 70 kDa monomers containing 644 residues. Each monomer is folded into two major domains, an N-terminal and a C-terminal domain, connected by a linker domain which is further divided into two smaller domains. The N-terminal domain is composed of an (α/β)8 TIM barrel where the catalytic site is located. The (α/β)8 TIM barrel has N-and C-terminal extensions which are highly important and involved in the dimerization of the protein. The active site is located at the C-terminal extension of the N-terminal domain, and binds the divalent cation Mn2+ and 2-ketoisovalerate. This metal-binding center has a pair of His residues in addition to an Asp residue. The presence of these amino acid residues allows for a lack of discrimination towards divalent metals that will be discussed later. The connecting linker domain is composed of two smaller domains, where one consists of an α-helix and two β-strands, while the other has three αhelices. The residues linking the two small subdomains of the linker domain are disordered and flexible. The C-terminus of MtIPMS consist of a regulatory domain composed of two identical βββα units, built as a three layer β-α-β sandwich 96.


Biochemistry

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Page 22 of 51

Figure 5. The 2.21 Å resolution three-dimensional structure of isopropylmalate synthase from M. tuberculosis (PDB code 3U6W). The homodimer is colored by monomer and the loops are displayed in yellow. A close-up view of the active site displaying α-ketoisovalerate and the metal binding center coordinated by the two His residues and Asp. The C-terminal regulatory domain of MtIPMS is the binding site for L-leucine and is responsible for the control of the activity of this enzyme

96

. IPMS is regulated via a product

inhibition mechanism by L-leucine in many organisms including M. tuberculosis. L-leucine was shown to be a reversible, slow-onset inhibitor of MtIPMS and its affinity for L-leucine is substrate-binding independent. The binding of L-leucine does not affect the quaternary structure of MtIPMS, however, binding of L-leucine results in an increase in the stability of both the Nand C-terminal protein domains. L-leucine acts as a V-type inhibitor by binding in a noncooperative fashion at the interface of the dimer approximately 50 Å distant from the active site 97-100

. MtIPMS displays modest activity with small keto acids substrates including pyruvate, 2-

ketobutyrate and 2-ketovalerate. However, the efficiency of the reaction with 2-ketoisovalerate is much higher

99

. The enzyme is extremely specific with regards to the acyl donor, with only 22 ACS Paragon Plus Environment

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Biochemistry

acetyl-CoA showing any activity. MtIPMS follows a non-rapid equilibrium random bi-bi kinetic mechanism 99. The chemical mechanism of the MtIPMS- catalyzed reaction consists of the enolization of acetyl-CoA, followed by an aldol condensation with a rapid protonation of the 2-hydroxyl group of 2-isopropylmalyl-CoA by an active site acid. The hydrolysis of the thioester carbon of the intermediate yields the formation of a tetrahedral intermediate whose breakdown releases the two products, 2-isopropylmalate and CoA (Scheme 5) 99.

Scheme 5. The chemical mechanism of isopropylmalate synthase. MtIPMS can use a broad range of divalent metals to catalyze its reaction, including Mg2+, Mn2+, Co2+, Ni2+ and Ca2+, with Mg2+ being the preferred metal. Metal binding induces contacts between the N-terminal catalytic domain and the C-terminal regulatory domain of MtIPMS, thus establishing structural cooperativity 101. Both Zn2+ and Cd2+ inhibit the enzyme

99

by inducing a

partial denaturation and/or unfolding of the domain 101. Monovalent cations also play an essential role in the activity of MtIPMS. The enzyme can utilize a large number of monovalent cations, however K+ and Rb+ are the preferred activators 99. There is no direct interaction of the K+ with the substrates 2-ketoisovalerate and acetyl-CoA, instead K+ plays an allosteric effector role and alters the surroundings of the Mg2+ binding-site without changing the enzyme structure

99, 101

.


Biochemistry

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Page 24 of 51

The rate-limiting step of the reaction catalyzed by MtIPMS in the absence of L-leucine has been proposed as being the release of products, however, upon L-leucine binding, the rate-limiting step shifts to the hydrolysis of the thioester carbon of the intermediate isopropylmalyl-CoA 98. The essentiality of this enzyme in bacteria, and it’s absence in mammals, makes IPMS a very interesting drug target to be exploited. So far, only in silico inhibition studies have been reported for the mycobacterial enzyme

102

. Therefore, the development of compounds targeting

IPMS remain to be explored.

6. LeuC/D- Isopropylmalate Isomerase The second exclusive enzyme in the branch of L-leucine biosynthesis is the leuCDencoded isopropylmalate isomerase (EC 4.2.1.33; IPMI) which catalyzes the isomerization of 2ispropylmalate to 3-isopropylmalate.

103, 104

. In some organisms, such as S. typhimurium and E.

coli, the genes encoding enzymes involved specifically in the leucine biosynthesis branch are organized in a single operon leuABCD and co-expressed. In 1981, S. typhimurium IMPI was identified as a multimeric enzyme formed by two separate genes, leuC and leuD 105. The enzyme was purified and the products of the leuC and leuD genes were present in a 1:1 ratio. The activity of S. typhimurium IPMI was analyzed in bacterial crude extracts, and the enzyme displayed very low activity. Attempts to purify the protein to homogeneity resulted in a complete loss in activity 105

. The chemical reaction catalyzed by IPMI presumably involves a base-assisted

dehydration of 2-isopropylmalate to generate a cis-vinylogous intermediate. The rotation of the intermediate in the active site followed by a trans addition of water results in the formation of the


Page 25 of 51

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Biochemistry

3-isopropylmalate product

106, 107

(Scheme 6). This reaction can be continuously measured by

observing the decrease in absorbance of citraconate, an isopropylmalate analogue, at 253 nm 108.

Scheme 6. The chemical mechanism of the isopropylmalate isomerase.

Due to the similarity of the reaction catalyzed by IPMI and the mitochondrial enzyme aconitase (ACN) 107, and sequence comparisons superfamily

110

109

, IPMI is known to be a member of the ACN

. ACNs are well characterized monomeric enzymes that requires an intact [4Fe-

4S] cluster for activity, catalyzing the isomerization of citrate to isocitrate with the production of the intermediate, cis-aconitate. Studies of an E. coli mutant strain lacking peroxidases including katG, katE and ahp, and treated with H2O2 found that supplementation of the media with 2ketoisocaproate rescued the growth of the bacteria in the presence of the peroxide. By employing a plasmid that overexpressed IPMI in the H2O2-treated culture, the growth defect was corrected, confirming the sensitivity of IPMI to reactive oxygen species

108

. IPMI inactivation by H2O2 is

due to the abstraction of an electron from the [4Fe-4S] cluster altering it to [4Fe-4S]3+, which is an unstable valence for the cluster, and leads to the release of Fe2+ which results in the inactivity of the remaining [3Fe-4S]+ cluster

108

. The oxygen sensitivity of [4Fe-4S] cluster enzymes such

as IPMI, makes it difficult to study since it requires an anaerobic environment.


Biochemistry

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Page 26 of 51

IPMIs are classified into two different groups depending on their subunit composition. The first group is composed by fungal IPMIs which are 80-90 kDa monomeric proteins, while in bacteria and archaea, the enzymes are heterodimers composed of a 45-50 kDa large leuC subunit and to a 15-20 kDa small leuD subunit. The IPMIs from the second group are only active when the two subunits come together to form the heterodimer. The large subunit presents three conserved Cys residues which are proposed ligands for the [4Fe-4S]3+ cluster, while 2isopropylmalate is predicted to bind the sequence motif GSSR, located on the small subunit To date, only one structure of the large subunit of IPMI has been solved and reported

112

111

.

. The

large subunit of the Methanococcus jannaschii (M. jannaschii) IPMI has been crystalized under aerobic conditions and the structure was solved at 1.8 Å resolution

112

. The monomer is

composed of 18 α-helices and 17 β-strands distributed in three domains organized in a triangular manner. The active site contains two disulfide bonds formed as a result of the oxidation of the presumed Cys ligands to the cluster, and is surrounded by the three domains of the large leuC subunit of M. jannaschii IPMI (Figure 6). No [4Fe-4S] cluster was present in the crystalized structure. A larger number of crystal structures have been reported for the small subunit of IPMI (PDB codes 3Q3W, 3H5E, 3H5H, 3H5J and 2HCU)

113

, including that of M. tuberculosis

111

(Figure 6).


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Biochemistry

Figure 6. Three-dimensional structure of: A) 1.8 Å resolution ilvC from M. jannaschii colored by domain. Mg2+ is represented as a green sphere (PDB code 4KP1); and B) 2.5 Å resolution ilvD from M. tuberculosis colored by secondary structure (β-sheets in light-magenta and αhelices in teal) (PDB code 3H5H).

M. tuberculosis IPMI (MtIPMI) has been purified to homogeneity as a homodimer (1:1 ratio) and the enzyme was more stable in solution than when each subunit was purified individually. When assayed, MtIPMI was completely inactive, probably due to the oxidation reaction leading to an inactive [3Fe-4S]+ cluster. Three variants of the MtIPMI leuD subunit, differing in the length of the protein, were crystalized at different resolutions. The overall fold of the small subunit is a twisting β/β/α- three-layered sandwich. Alignments of the MtIPMI leuD subunit with other homologues and a portion of aconitase led to the proposal that MtIPMI leuD Arg32 (ACN Arg580) plays an important role in substrate recognition by making important hydrogen bonds with the γ-carboxylate of 2-ispropylmalate. Alignment of MtIPMI leuC with mitochondrial ACN revealed that the enzymes share 28 and 43% sequence identity and


Biochemistry

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Page 28 of 51

similarity, respectively. The residues involved in binding of substrates, catalysis and cofactor coordination are all well conserved between the two enzymes. Therefore, it is likely that the heterodimer complex of MtIPMI is structurally similar to that of ACN. The importance of the leuCD genes and therefore of IPMI in M. tuberculosis growth and virulence has been recognized for over two decades. Disruption of the leuD gene in the Mycobacterium bovis (M. bovis) strain (BCG) causes a growth and infection impairment in mouse models 114. The auxotrophic profile is attributed to the inability of BCG ∆leuD to replicate and grow inside macrophages, since the bacteria cannot scavenge intracellular leucine

115

. The

same profile was observed when the leuD gene was disrupted in M. tuberculosis, and the successfully attenuated strain was also protective of mice challenged with a virulent strain of the bacilli. A double auxotroph strain (∆panCD∆leuCD) was even more protective than the ∆leuDalone, resulting in the protection of macaques co-infected with TB/SIV (simian immunodeficiency). These data support the essentiality of IMPI in the growth and survival of M. tuberculosis and confirms the potential of this enzyme as a potential drug target.

7. LeuB- Isopropylmalate Dehydrogenase The leuB-encoded isopropylmalate dehydrogenase (EC 1.1.1.85; IPMDH) is responsible for converting 3-isopropylmalate to 2-ketoisocaproate via oxidation of the second alcohol and decarboxylation. This reaction is NAD+-dependent and requires the presence of a divalent metal such as Mg2+ or Mn2+ and the monovalent cation K+ for activation

116-118

. IPMDH was first

purified and characterized in S. typhimurium in 1969 119. Starting with 19 g of crude extract, four purification steps yielded 120 mg of greater than 95% pure enzyme. S. typhimurium IPMDH is a 70 kDa homodimer in solution

119

. The optimum pH found for this enzyme was pH 9 and the


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Biochemistry

activity of S. typhimurium IPMDH was measured using a continuous spectrophotometric assay where the increase in absorbance at 340 nm due to NAD+ reduction was observed over time 119. The enzyme followed Michaelis-Menten kinetics for both substrates. Crystal structures of IPMDH from a number of different organisms have been solved 120125

including M. tuberculosis 126. IPMDH from E. coli and S. typhimurium share 94.5 % sequence

identity, while these enzymes share 51% sequence identity with the Thermus thermofilus (T. thermophilus) IPMDH 125. The overall topology of all three proteins is however very similar 125. Most IMPDHs are homodimeric proteins and the structure is generally composed of two (α/β) domains organized in a ten-stranded β-sheet where each monomer consists of 300-400 amino acid residues. The crystal structure of M. tuberculosis IPMDH (MtIPMDH) was solved at 1.65 Å resolution in the absence of any bound substrates or cofactors, and in solution it is a 70 kDa homodimer (Figure 7). The enzyme is approximately 40 % sequence identical to other bacterial orthologues 126.


Biochemistry

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Page 30 of 51

Figure 7. The 1.65 Å resolution three-dimensional structure of the asymmetric unit of isopropylmalate dehydrogenase from M. tuberculosis (PDB code 1W0D). The tetrameric enzyme is colored by monomer.

Some of the structurally characterized IPMDHs display different conformations, varying from open to partially closed and fully closed active sites. The superimposition of MtIPMDH with the E. coli, S. typhimurium and T. thermophilus structures revealed that MtIPMDH presents a closed conformation, however the open conformation can also be observed in some of the subunits of this enzyme

126

. The active site of MtIPMDH was inferred to be located at the cleft

between the two domains of the enzyme, based on a sequence alignment with the previously characterized IPMDH from Thiobacillus ferrooxidans of isopropylmalate

126

121

which was crystalized in the presence

. The amino acids involved in isopropylmalate and Mg2+ binding are

conserved between the two structures

126

. A similar alignment strategy was used to compare the

NAD+-binding site of MtIPMDH with T. thermophilus IPMDH 120.


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Biochemistry

IPMDH is a member of the metal ion-dependent, β-hydroxyacid oxidative decarboxylase family. Enzymes such as malic enzyme and isocitrate dehydrogenase are also part of this family 127

. In general, these enzymes catalyze a reaction consisting of a pyridine nucleotide-dependent

reversible secondary alcohol oxidation followed by an irreversible decarboxylation that leads to the formation of an enol-intermediate; which is tautomerized and results in the final product, which in the case of IPMDH is 2-ketoisocaproate 117, 127 (Scheme 7).

Scheme 7. The chemical mechanism of isopropylmalate dehydrogenase.

IPMDH follows a random steady-state kinetic mechanism in common with other members of the β-hydroxy-acid oxidative decarboxylase family 116, 127. Transient kinetic studies proposed that in T. thermophilus IPMDH

116

, NAD+ binds rapidly, whereas the binding of

isopropylmalate is slower and induces a conformational change that brings the protein in to a closed-state. According to their studies, the closed conformation is a requirement and determines the rate of isopropylmalate oxidation and formation of NADH (reduced nicotinamide adenine dinucleotide) in the pre-steady-state. The decarboxylation and the tautomerization processes are spontaneous and occur faster and without conformational changes. However, product release requires the opening of the protein domains, and is the rate-limiting step in catalytic turnover 116. Structural studies and quantum mechanics/molecular mechanics (QM/MM) calculations, resulted in the following proposed mechanism for the T. thermophilus IPMDH. A general base, Lys185, 31 ACS Paragon Plus Environment

Biochemistry

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Page 32 of 51

deprotonates the OH group of isopropylmalate via a water molecule. The oxidation of isopropylmalate and hydride transfer display a higher energy barrier and therefore are the ratelimiting step in this model. However, K+ can increase the rate of hydride transfer by 1000-2000fold. Decarboxylation is a spontaneous step, and has a very low energy barrier and drives the formation of the final products

117

. In this case, the hydride transfer step seems to account for

rate-limitation. IPMDH also has potential for herbicidal and antibacterial drug development. Oisobutenyl oxalylhydroxamate (O-IbOHA) is a compound initially designed to display increased herbicidal activity against KARI enzymes 118. However, through supplementation studies with Lleucine and 2-ketoisocaproate, IPMDH was found to be the target of this compound in plants and in S. typhimurium

118

. Besides O-IbOHA, several other inhibitors have been designed and

assayed against the T. thermophilus enzyme. In general, these compounds act as competitive inhibitors and display inhibition constants in the nanomolar range

118, 128, 129

. To date, no

experimental data has been published on the activity of O-IbOHA against MtIPMDH. However, small molecule docking of the inhibitor identified that the potential binding site of O-IbOHA in MtIPMDH is very likely to be the same binding site as isopropylmalate. O-IbOHA likely inhibits MtIPMDH by mimicking the enol-intermediate of the reaction

126

. This data can be used as a

foundation for future design of MtIPMDH-inhibitors with potential anti-mycobacterial activity.

8. IlvE- Branched-Chain Aminotransferase The final step in the synthesis of all three BCAAs involves the transfer of the α-amino group of L-glutamate to the α-carbon of 2-ketoisocaproate, 2-ketoisovalerate and 2-keto-3methylvalerate, the keto acid precursors of L-leucine, L-valine and L-isoleucine, respectively.


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Biochemistry

Transamination reactions were first identified in the late 1930’s and observed to be biologically relevant in many organisms

130

. The first aminotransferases to be discovered were L-glutamate

aminotransferase and L-aspartate aminotransferase

130

. E. E. Snell, who had previously

characterized chemical transaminations in the presence of pyridoxal, made the association that the enzymatically-catalyzed transamination reactions were dependent on the presence of this cofactor

131

. Gunsalus reported in 1950 that bacteria have a variety of distinct transaminases and

confirmed the need for pyridoxal as a co-factor/coenzyme for enzyme activity 132. The ilvE-encoded branched-chain aminotransferase (EC 2.6.2.41; BCAT) was first isolated in 1952 from Escherichia coli

133

, and two different aminotransferases were identified.

The first one had higher activity in the presence of aromatic amino acids and was named Transaminase A, and the second, Transaminase B, displayed higher activity in the presence of the BCAA’s isoleucine, leucine and valine 133. Between the 1950’s and 1976, studies on BCATs from several Gram-negative bacteria were reported. These studies are covered in a review on BCAAs catabolism in bacteria

134

. We will rather examine the anabolic reaction, or BCAAs

synthesis, as well as what is known of BCATs in terms of mechanism, structures and its potential for drug development. In the anabolic direction, BCATs are responsible for the transfer of an amino group from L-glutamate to the α-keto acid form of the respective amino acid to be synthesized

135

. This

reaction is reversible and depends on the coenzyme PLP being covalently bound to the enzyme through a Schiff base with a protein lysine residue 136. The first three-dimensional structure of a bacterial BCAT was reported in 1997

137

. The homohexameric E. coli structure was reported at

2.5 Å resolution and displayed an interesting triangular prism shape arranged as a doublet of trimers. Numerous structures of BCATs from different eubacteria, as well as human BCATs,


Biochemistry

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Page 34 of 51

have been solved, including the M. tuberculosis enzyme (MtIlvE) and all belong to the type IV fold class of PLP-dependent transaminases

136

. This classification is due to the presence of two

domains connected by an interdomain loop, and where the chemical reaction occurs at the re face of the Schiff base to the PLP cofactor

136

. The MtIlvE structure was solved at 1.9 Å resolution

and is a homodimer of 40 kDa with a the pyridoxamine 5’-phosphate (PMP) molecule present at the active site of both monomers

136

. Each monomer is composed of two domains that together

form the active-site. The first domain is composed of a core eight β-strands surrounded by three α-helices and the second domain consists of two β-sheets surrounded by three α-helices. A particular Cys residue is found in MtIlve and absent in any other orthologue. In this crystal structure, the thiol groups of Cys196 are 3.6 Å apart and therefore do not form a disulfide bond. BCATs, as well as other aminotransferases, display a ping-pong kinetic mechanism. In the ping half-reaction, the α-amino group from the donor amino acid reacts with the Schiff base PLP form of the enzyme, and then chemistry takes place followed by an α-keto acid release. The enzyme is now in the PMP form and the second half-reaction can take place. The pong halfreaction starts with the binding of a different α-keto acid to the PMP form of the enzyme, followed by proton transfer from the C4’ of the cofactor to the carbon of the ketimine to generate the new BCAA and regenerate the enzyme in its PLP-form. The detailed chemical mechanism of the M. tuberculosis BCAT 138 revealed that transamination occurs via an unusual 1,3-prototropic shift mechanism

138

, where α-C-H bond cleavage from L-glutamate occurs simultaneously with

the protonation of the C4’ of the PLP cofactor in the same transition state (Scheme 8).


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Biochemistry

Scheme 8. The chemical mechanism of IlvE from M. tuberculosis.

The absence of an observable quininoid intermediate, which is a common feature of PLPdependent enzyme reactions

139, 140

, confirmed the concerted mechanism of MtIlvE. Some PLP-

dependent enzymes have reported ketamine hydrolysis as the rate-limiting step of the reaction 141, 142

. Despite kinetic, chemical and structural similarities, BCATs from bacteria exhibit

different substrate specificities. The E. coli BCAT and MtIlvE can utilize a large number of amino acids substrates, ranging from BCAAs to methionine and aromatic amino acids 138, 143. On the other hand, the mammalian isoforms have no activity in the presence of aromatic amino acids and are active with aspartate

144

. These small variances play an important role in selectivity and

therefore can be used for rational drug design. The potential of this enzyme as a drug target has been evaluated for many years in humans due to its selective inhibition by gabapentin, used in the treatment of epilepsy 144-146. The two human BCAT isoforms (cytosolic BCAT and mitochondrial BCAT) share 58% sequence identity, yet, gabapentin does not inhibit the mitochondrial isoform. MtIlvE shares 31% sequence identity with the mitochondrial BCAT, and the tuberculosis enzyme is not inhibited by gabapentin

136, 143

, showing that MtIlvE has the potential to be specifically targeted. Inhibition

studies of MtIlvE with aminooxy compounds were performed and demonstrated the druggability of this enzyme 143. The best mycobacterial inhibitor was O-allylhydroxylamine, which displayed a KI of 22 µM and a MIC value of 156 µM. The difference between the O-allylhydroxylamine 35 ACS Paragon Plus Environment

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MIC value in M. tuberculosis and the KI for MtIlvE may be the result of inhibitory effects of the compound on multiple PLP-dependent enzymes. A similar feature was observed when MtIlvE was inhibited by D- and L-cycloserine

135

. D-cycloserine is a second-line drug used in the

treatment of MDR-TB and inhibits two sequential reactions of the peptidoglycan biosynthetic pathway: the PLP-dependent alanine racemase and D-alanine-D-alanine ligase

147

. MtIlvE is

inhibited by both cycloserine isomers in a time- and concentration- dependent fashion. Lcycloserine is however a 40-fold more potent inhibitor of the enzyme in comparison to Dcycloserine, displaying a KI of 88 µM. The MIC values also show a 10-fold better inhibitory effect with the L-isomer and in all cases supplementation with BCAAs either individually or in combination, did not rescue the growth of the bacteria. It was suggested that the cycloserine isomers, as well as the aminooxy compounds, are generalized inhibitors of PLP-dependent enzymes 135. The mechanism of inactivation of MtIlvE by the cycloserine isomers was shown to be the result of the aromatization of the cycloserine ring to form a stable PLP adduct

135

. The

structure of the inhibited MtIlvE-D-cycloserine complex was solved at 1.7 Å resolution and reveals an intact and planar D-cycloserine ring (Figure 8).


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Figure 8. The 1.7 Å resolution three-dimensional structure of the branched-chain aminotransferase IlvE from M. tuberculosis (PDB code 5U3F). The homodimeric protein is colored by secondary structure: β- sheets (light-magenta) and α- helices (teal). Both monomers contain the bound-irreversible complex PMP-D-cycloserine at the active site.

CONCLUSIONS The enzymes belonging to the BCAAs biosynthetic pathway in bacteria are an excellent potential source of targets to be explored for the development of new antibacterial agents. Of particular interest, in M. tuberculosis, all the enzymes in this pathway are essential for growth and survival of the bacilli and the disruption of any enzyme of the pathway may have a serious consequence for the survival of the bacteria. M. tuberculosis is relatively poor at scavenging BCAAs from the host cell and therefore, as observed for many enzymes of the BCAA pathway, it can be used as a strategy for the development of auxotrophic strains to be used as vaccines or drug inhibition candidates. In addition, the advantages of targeting this pathway in bacteria is evident, due to the lack of a similar pathway in mammals (only BCATs are present in mammals), 37 ACS Paragon Plus Environment

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which would reduce related toxicity. Several compounds, including herbicides, have been identified as potent inhibitors of the enzymes from this pathway in different organisms. Leading compounds can be found through the screening of the already existing molecules, or used as a foundation for better and more specific inhibitors. In addition, depending on the enzyme targeted, not only BCAAs synthesis is affected, since some of the products of this pathway, such as α-ketoisovalerate, are precursors for the pantothenate biosynthetic pathway. The inhibition of AHAS, KARI, DHAD and BCAT in bacteria affects the synthesis and/or recycling of essential amino acids and metabolites (BCAAS, methionine pantothenate and CoA) which can be explored as a “death by a thousand cuts” strategy against pathogenic organisms. In summary, the enzymes of the BCAA biosynthetic pathway in pathogenic bacteria seem to represent mechanistically and structurally well-characterized targets for further exploitation for antibacterial drug design.


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AUTHOR INFORMATION Corresponding Author: * E-mail: [email protected] ORCID: John S. Blanchard: 0000-0002-9195-4402 Funding Sources: This work was supported by a grant (NIH AI060899) to J.S.B and Science Without Boarders fellowship - CAPES, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil - to T.M.A.F. Notes: The authors declare no conflicts of interest with the contents present in this article. ABBREVIATIONS: ACN, aconitase; AHAS, acetohydroxyacid synthase; AHAThDP, acetohydroxyacid-thiamin diphosphate; BCAA, branched-chain amino acid; BCAT, branched-chain aminotransferase; BCG, Bacille Balmette-Guerin; Coa, coenzyme A; DHAH, dihydroxyacid dehydratase; DNA, deoxyribonucleic acid; FAD, flavine adenine dinucleotide; HEThDP, hydroxyethylthiamin diphosphate; IPMDH, isopropylmalate dehydrogenase; IPMI, isopropylmalate isomerase; IpOHA, N-isopropyl oxalylhydroxamate; IPMS, isopropylmalate synthase; KARI, keto/ketolacid isomeroreductase; LThDP, 2-lactyl-thiamin diphosphate; MDR, multidrug-resistant, MIC, minimal inhibitory concentration; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adeninde nucleotide phosphate; NO, nitric oxide; O-IbOHA, O-isobutenyl oxalylhydroxamate; PDB, protein data bank; PLP, pyridoxal 5’-phosphate; PMP, pyridoxamine 5’-phosphate, PO, pyruvate oxidase; QM/MM, quantum mechanics/molecular mechanics; SU,


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sulfonylurea; TB, tuberculosis; TD, threonine dehydratase; ThDP, thiamin diphosphate; XDR, extensively drug-resistant.


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Biochemistry

BCAA Biosynthetic Pathway Threonine

IlvA

2-ketobutyrate

ilvBN

ilvBN 2-aceto-2-hydroxybutyrate

2,3-dihydroxy-3-methylvalerate

2-isopropylmalate leuCD

leuA

2, 3-dihydroxyisovalerate

3-isopropylmalate

ilvD

ilvD 2-keto-methylvalerate

Isoleucine

2-acetolactate ilvC

ilvC

ilvE

Pyruvate

leuB 2-ketoisocaproate

2-ketoisovalerate ilvE

lvE

Valine

Leucine

Pantothenate ACS Paragon Plus Environment

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures

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