Isoniazid and thioacetazone may exhibit antitubercular activity by binding directly with the active site of mycolic acid cyclopropane synthase: Hypothesis based on computational analysis

Isoniazid and thioacetazone are the two important antitubercular drugs. In case of thioacetazone it is established that it inhibits mycolic acid cyclopropane synthase but the exact binding site accounting for such inhibition is presently unknown. In case of isoniazid its action on the said enzyme is unexplored. In this work we have analyzed the binding of isoniazid and thioacetazone with mycolic acid cyclopropane synthase (CmaA1 and CmaA2) using tools of computational biology. We have observed that thioacetazone fits well at the active site of CmaA1 and CmaA2 while isoniazid binds at the active site of CmaA1 only. We have recommended experimental validation of such results. If such results are proved to be fact it will explore the exact binding site of thioacetazone and discover a new mechanism of anti-tubercular action of isoniazid.     

Further insight into S-adenosylmethionine-dependent methyltransferases: structural characterization of Hma, an enzyme essential for the biosynthesis of oxygenated mycolic acids in Mycobacterium tuberculosis

Mycolic acids are major and specific components of the cell envelope of Mycobacteria that include Mycobacterium tuberculosis, the causative agent of tuberculosis. Their metabolism is the target of the most efficient antitubercular drug currently used in therapy, and the enzymes that are involved in the production of mycolic acids represent important targets for the development of new drugs effective against multidrug-resistant strains. Among these are the S-adenosylmethionine-dependent methyltransferases (SAM-MTs) that catalyze the introduction of key chemical modifications in defined positions of mycolic acids. Some of these subtle structural variations are known to be crucial for both the virulence of the tubercle bacillus and the permeability of the mycobacterial cell envelope. We report here the structural characterization of the enzyme Hma (MmaA4), a SAM-MT that is unique in catalyzing the introduction of a methyl branch together with an adjacent hydroxyl group essential for the formation of both keto- and methoxymycolates in M. tuberculosis. Despite the high propensity of Hma to proteolytic degradation, the enzyme was produced and crystallized, and its three-dimensional structure in the apoform and in complex with S-adenosylmethionine was solved to about 2 A. Thestructuresshowtheimportantroleplayedbythemodificationsfound within mycolic acid SAM-MTs, especially thealpha2-alpha3 motif and the chemical environment of the active site. Essential information with respect to cofactor and substrate binding, selectivity and specificity, and about the mechanism of catalytic reaction were derived.     

Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria

Background

Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets.

Methodology/Principle Findings

We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the α- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs.

Conclusions/Significance

This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.     

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA,a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60–90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C_38–42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors.     

Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity

This was the first study that achieved a narrowing of the substrate specificity of water soluble glucose dehydrogenase harboring pyrroloquinoline quinone as their prosthetic group, PQQGDH-B. We conducted the introduction of amino acid substitutions into the loop 6BC region of the enzyme, which made up the active site cleft without directly interacting with the substrate, and constructed a series of site directed mutants. Among these mutants, Asn452Thr showed the least narrowed substrate specificity while retaining a similar catalytic efficiency, thermal stability and EDTA tolerance as the wild-type enzyme. The relative activities of mutant enzyme with lactose were lower than that of the wild-type enzyme. The altered substrate specificity profile of the mutant enzyme was found to be mainly due to increase in Km value for substrate than glucose. The predicted 3D structures of Asn452Thr and the wild-type enzyme indicated that the most significant impact of the amino acid substitution was observed in the interaction between the 6BC loop region with lactose.     

Mycolic acid synthesis by Mycobacterium aurum cell-free extracts

The first cell-free system capable of synthesizing whole mycolic acids: (R1CH(OH)CH(R2)COOH, with 60 to 90 carbon atoms) from [1-14C]acetate is described and preliminary investigations into some of its requirements and properties are reported. Biosynthetic activity for mycolic acids occurred in an insoluble fraction (40 000 X g pellet) from disrupted cells of Mycobacterium aurum (ATCC 23366-type strain); it produced mycolic acids, but a very small amount of non-hydroxylated fatty acids. The predominant product was unsaturated mycolic acid (type I), while oxo- (type IV) and dicarboxy- (type VI) mycolic acids were synthesized to a lesser extent. When [1-14C]palmitic acid was used as a marker, no labelled mycolic acid was detected. The reaction required a divalent cation (Mg2+ or Mn2+), KHCO3 and O2. Neither CoA, NADH, NADPH nor ATP were necessary, but CoA rather increased the synthesis of non-hydroxylated fatty acids. Glucose or trehalose were not required. Avidin inhibited the biosynthesis of the three types of mycolic acid indicating the presence of a biotin-requiring enzyme in the reaction sequence and therefore a carboxylation step, but citrate had no allosteric effect. Iodoacetamide inhibited the system. These first data are in favor of a complex multienzyme system.     

Crystal structure of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III

Mycolic acids (alpha-alkyl-beta-hydroxy long chain fatty acids) cover the surface of mycobacteria, and inhibition of their biosynthesis is an established mechanism of action for several key front-line anti-tuberculosis drugs. In mycobacteria, long chain acyl-CoA products (C(14)-C(26)) generated by a type I fatty-acid synthase can be used directly for the alpha-branch of mycolic acid or can be extended by a type II fatty-acid synthase to make the meromycolic acid (C(50)-C(56)))-derived component. An unusual Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) has been identified, purified, and shown to catalyze a Claisen-type condensation between long chain acyl-CoA substrates such as myristoyl-CoA (C(14)) and malonyl-ACP. This enzyme, presumed to play a key role in initiating meromycolic acid biosynthesis, was crystallized, and its structure was determined at 2.1-A resolution. The mtFabH homodimer is closely similar in topology and active-site structure to Escherichia coli FabH (ecFabH), with a CoA/malonyl-ACP-binding channel leading from the enzyme surface to the buried active-site cysteine residue. Unlike ecFabH, mtFabH contains a second hydrophobic channel leading from the active site. In the ecFabH structure, this channel is blocked by a phenylalanine residue, which constrains specificity to acetyl-CoA, whereas in mtFabH, this residue is a threonine, which permits binding of longer acyl chains. This same channel in mtFabH is capped by an alpha-helix formed adjacent to a 4-amino acid sequence insertion, which limits bound acyl chain length to 16 carbons. These observations offer a molecular basis for understanding the unusual substrate specificity of mtFabH and its probable role in regulating the biosynthesis of the two different length acyl chains required for generation of mycolic acids. This mtFabH presents a new target for structure-based design of novel antimycobacterial agents.     

Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity

This was the first study that achieved a narrowing of the substrate specificity of water soluble glucose dehydrogenase harboring pyrroloquinoline quinone as their prosthetic group, PQQGDH-B. We conducted the introduction of amino acid substitutions into the loop 6BC region of the enzyme, which made up the active site cleft without directly interacting with the substrate, and constructed a series of site directed mutants. Among these mutants, Asn452Thr showed the least narrowed substrate specificity while retaining a similar catalytic efficiency, thermal stability and EDTA tolerance as the wild-type enzyme. The relative activities of mutant enzyme with lactose were lower than that of the wild-type enzyme. The altered substrate specificity profile of the mutant enzyme was found to be mainly due to increase in Km value for substrate than glucose. The predicted 3D structures of Asn452Thr and the wild-type enzyme indicated that the most significant impact of the amino acid substitution was observed in the interaction between the 6BC loop region with lactose.     

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment

In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3',5'-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotide binding. The single ion-occupied, nucleotide-bound state defines a novel, open adenylyl cyclase state. In contrast, bicarbonate increases the catalytic rate by inducing marked active site closure and recruiting a second, catalytic ion. The phosphates of the bound substrate analogs are rearranged, which would facilitate product formation and release. The mechanisms of calcium and bicarbonate sensing define a reaction pathway involving active site closure and metal recruitment that may be universal for class III cyclases.     

Crystal structures of nonoxidative zinc-dependent 2,6-dihydroxybenzoate (gamma-resorcylate) decarboxylase from Rhizobium sp. strain MTP-10005

Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one (alphabeta)8 triose-phosphate isomerase-barrel domain with three functional linkers and one C-terminal tail. The native enzyme possesses one Zn2+ ion liganded by Glu8, His10, His164, Asp287, and a water molecule at the active site center, although the enzyme has been reported to require no cofactor for its catalysis. The substrate carboxylate takes the place of the water molecule and is coordinated to the Zn2+ ion. The 2-hydroxy group of the substrate is hydrogen-bonded to Asp287, which forms a triad together with His218 and Glu221 and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn2+-dependent decarboxylation.     

Structural and biochemical studies on the regulation of biotin carboxylase by substrate inhibition and dimerization

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO₂ donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 Å resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca²⁺ ions or two ADP molecules and one Mg²⁺ ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca²⁺ ion and the Mg²⁺ ion are associated with the ADP molecule in the active site, and the other Ca²⁺ ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.     

Characterization of CmaA, an adenylation-thiolation didomain enzyme involved in the biosynthesis of coronatine

Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-(32)PP(i) exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and L-[G-(3)H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.     

Mechanism of mycolic acid cyclopropane synthase: a theoretical study

The reaction mechanism of mycolic acid cyclopropane synthase is investigated using hybrid density functional theory. The direct methylation mechanism is examined with a large model of the active site constructed on the basis of the crystal structure of the native enzyme. The important active site residue Glu140 is modeled in both ionized and neutral forms. We demonstrate that the reaction starts via the transfer of a methyl to the substrate double bond, followed by the transfer of a proton from the methyl cation to the bicarbonate present in the active site. The first step is calculated to be rate-limiting, in agreement with experimental kinetic results. The protonation state of Glu140 has a rather weak influence on the reaction energetics. In addition to the natural reaction, a possible side reaction, namely a carbocation rearrangement, is also considered and is shown to have a low barrier. Finally, the energetics for the sulfur ylide proposal, which has already been ruled out, is also estimated, showing a large energetic penalty for ylide formation.     

Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site

Tobacco etch virus (TEV) protease is a cysteine protease exhibiting stringent sequence specificity. The enzyme is widely used in biotechnology for the removal of the affinity tags from recombinant fusion proteins. Crystal structures of two TEV protease mutants as complexes with a substrate and a product peptide provided the first insight into the mechanism of substrate specificity of this enzyme. We now report a 2.7A crystal structure of a full-length inactive C151A mutant protein crystallised in the absence of peptide. The structure reveals the C terminus of the protease bound to the active site. In addition, we determined dissociation constants of TEV protease substrate and product peptides using isothermal titration calorimetry for various forms of this enzyme. Data suggest that TEV protease could be inhibited by the peptide product of autolysis. Separate modes of recognition for native substrates and the site of TEV protease self-cleavage are proposed.     

Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors

Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2’,4,4’-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.     

Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase

Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug-an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme.     

Purification and substrate specificity of bovine angiotensin-converting enzyme

Angiotensin-converting enzyme was solubilized from bovine lung with detergent and purified over 2300-fold to physical homogeneity by a combination of ammonium sulfate fractionation, molecular sieve chromatography, and ion exchange chromatography. The purified enzyme had an apparent molecular weight of 126,000 in both the denatured, and reduced, denatured forms as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 13.6 units/mg. It was inhibited by EDTA and activated by chloride ion. Chloride functioned as a nonessential activator by raising the Vmax 4.26-fold and lowering the KM 5.99-fold under saturating conditions. Under these conditions, the Vmax was 1.2 mumol/min/unit and the KM was 1.3 mM. Three series of peptides having the general structures, Hip-His-X, Hip-X-Leu, and Hip-X-His-Leu were synthesized and used to examine the binding specificity and substrate specificity of the enzyme for amino acids in the COOH-terminal (P'2), penultimate COOH-terminal (P'1), and antepenultimate COOH terminal (P1) peptide positions. These studies indicated that in terms of binding specificity, the relative importance of these three positions was P'2 > P'1 > P1, while the reverse order P1 > P'1 > P'2 was observed for the relative contribution to substrate specificity. Three peptides, Hip-His-D-Leu, Hip-D-His-Leu, and Hip-D-Phe-His-Leu, were also synthesized and used to examine the stereochemical requirements of the enzyme in terms of both peptide binding and hydrolysis. Hydrolysis was found to require an L amino acid in all three positions. In contrast, all three peptides bound to the enzyme.     

Organization of an efficient carbonic anhydrase: implications for the mechanism based on structure-function studies of a T199P/C206S mutant

Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)(1) reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and beta-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The beta-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.     

The specificity of methyl transferases involved in trans mycolic acid biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Trans mycolic acid content is directly related to cell wall fluidity and permeability in mycobacteria. Carbon-13 NMR spectroscopy of mycolic acids isolated from Mycobacterium tuberculosis (MTB) and Mycobacterium smegmatis (MSM) fed 13C-labeled precursor molecules was used to probe the biosynthetic pathways that modify mycolic acids. Heteronuclear correlation spectroscopy (HMQC) of ketomycolic acid from MTB allowed assignment of the complete 13C-NMR spectrum. Incorporation patterns from [1-13C]-acetate and [2-13C]-acetate feeding experiments suggested that the mero chain and alpha branch of mycolic acids are both synthesized by standard fatty acid biosynthetic reactions. [13C-methyl]-L-methionine was used to specifically label carbon atoms derived from the action of the methyl transferases involved in meromycolate modification. To enrich for trans mycolic acids a strain of MTB overexpressing the mma1 gene was labeled. Carbon-carbon coupling was observed in mycolate samples doubly labeled with 13C-acetate and [13C-methyl]-L-methionine and this information was used to assess positional specificity of methyl transfer. In MTB such methyl groups were found to occur exclusively on carbons derived from the 2 position of acetate, while in MSM they occurred only on carbons derived from the 1 position. These results suggest that the MSM methyltransferase MMAS-1 operates in an inverted manner to that of MTB.     

X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: insight into the role of calcium and surrounding amino acids in active site geometry and catalysis

Phosphatidylinositol-specific phospholipase Cs (PLCs) are a family of phosphodiesterases that catalyze the cleavage of the P-O bond via transesterification using the internal hydroxyl group of the substrate as a nucleophile, generating the five-membered cyclic inositol phosphate as an intermediate or product. To better understand the role of calcium in the catalytic mechanism of PLCs, we have determined the X-ray crystal structure of an engineered PLC enzyme from Bacillus thuringiensis to 2.1 A resolution. The active site of this enzyme has been altered by substituting the catalytic arginine with an aspartate at position 69 (R69D). This single-amino acid substitution converted a metal-independent, low-molecular weight enzyme into a metal ion-dependent bacterial PLC with an active site architecture similar to that of the larger metal ion-dependent mammalian PLC. The Ca(2+) ion shows a distorted square planar geometry in the active site that allows for efficient substrate binding and transition state stabilization during catalysis. Additional changes in the positions of the catalytic general acid/general base (GA/GB) were also observed, indicating the interrelation of the intricate hydrogen bonding network involved in stabilizing the active site amino acids. The functional information provided by this X-ray structure now allows for a better understanding of the catalytic mechanism, including stereochemical effects and substrate interactions, which facilitates better inhibitor design and sheds light on the possibilities of understanding how protein evolution might have occurred across this enzyme family.