Search

Recent evidence established that the cell envelope of Mycobacterium tuberculosis, the bacillus causing tuberculosis (TB), is coated by an α-glucan-containing capsule that has been implicated in persistence in a mouse infection model. As one of three known metabolic routes to α-glucan in mycobacteria, the cytoplasmic GlgE-pathway converts trehalose to α(1 → 4),α(1 → 6)-linked glucan in 4 steps. Whether individual reaction steps, catalyzed by trehalose synthase TreS, maltokinase Pep2, and glycosyltransferases GlgE and GlgB, occur independently or in a coordinated fashion is not known. Here, we report the crystal structure of M. tuberculosis TreS, and show by small-angle X-ray scattering and analytical ultracentrifugation that TreS forms tetramers in solution. Together with Pep2, TreS forms a hetero-octameric complex, and we demonstrate that complex formation markedly accelerates maltokinase activity of Pep2. Thus, complex formation may act as part of a regulatory mechanism of the GlgE pathway, which overall must avoid accumulation of toxic pathway intermediates, such as maltose-1-phosphate, and optimize the use of scarce nutrients.

The meso (or D,L) isomer of diaminopimelic acid (DAP), a precursor of L-lysine, is a key component of the pentapeptide linker in bacterial peptidoglycan. While the peptidoglycan incorporated in the highly complex cell wall of the pathogen Mycobacterium tuberculosis structurally resembles that of Escherichia coli, it is unique in that it can contain penicillin-resistant meso-DAP--meso-DAP linkages. The interconversion of L,L-DAP and meso-DAP is catalysed by the DAP epimerase DapF, a gene product that is essential in M. tuberculosis. Here, the crystal structure of the ligand-free form of M. tuberculosis DapF (MtDapF) refined to a resolution of 2.6 A is reported. MtDapF shows small if distinct deviations in secondary structure from the two-domain alpha/beta-fold of the known structures of Haemophilus influenzae DapF and Bacillus anthracis DapF, which are in line with its low sequence identity (or=27%) to the former. Modelling the present structure onto that of L,L-aziridino-DAP-bound H. influenzae DapF illustrates that a rigid-body movement of domain II and a rearrangement of the B4-A2 loop (residues 80-90) of domain I are likely to accompany the transition from the present inactive form to a catalytically competent enzyme. Despite a highly conserved active-site architecture, the model indicates that stabilization of the DAP backbone occurs in MtDapF through a tyrosine residue that is specific to mycobacterial DAP epimerases.

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.

Tuberculosis (TB) remains a leading cause of mortality worldwide. With the emergence of multidrug resistant TB, extensively drug resistant TB and HIV-associated TB it is imperative that new drug targets be identified. The potential of Mycobacterium tuberculosis inosine monophosphate dehydrogenase (IMPDH) as a novel drug target was explored in the present study. IMPDH exclusively catalyzes the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) in the presence of the cofactor nicotinamide adenine dinucleotide (NAD(+)). Although the enzyme is a dehydrogenase, the enzyme does not catalyze the reverse reaction i.e. the conversion of XMP to IMP. Unlike other bacteria, M. tuberculosis harbors three IMPDH-like genes, designated as Mt-guaB1, Mt-guaB2 and Mt-guaB3 respectively. Of the three putative IMPDH's, we previously confirmed that Mt-GuaB2 was the only functional ortholog by characterizing the enzyme kinetically. Using an in silico approach based on designed scaffolds, a series of novel classes of inhibitors was identified. The inhibitors possess good activity against M. tuberculosis with MIC values in the range of 0.4 to 11.4 µg mL(-1). Among the identified ligands, two inhibitors have nanomolar K(i)s against the Mt-GuaB2 enzyme.

Because of an increased emergence of resistance to current antitubercular drugs, there is a need for new antitubercular agents directed against novel targets. Diaminopimelic acid (DAP) biosynthetic enzymes are unique to bacteria and are absent in mammals and provide a rich source of essential targets for antitubercular chemotherapy. Herein, we review the structure and function of the mycobacterial DAP biosynthetic enzymes.

Peptidoglycan, the major structural component of the bacterial cell wall, is essential for bacterial growth. Since the polymer is absent in humans, disruption of peptidoglycan synthesis is an attractive method for eliminating bacteria in a search for new antibacterial agents. Peptidoglycan is a macromolecular polymer of a repeating disaccharide-peptide unit, where the peptide chains attached to adjacent sugar molecules are cross-linked. In the terminal stages of its synthesis, at the periplasm, the disaccharide units are polymerized by the transglycosylase (TG) enzyme, and the peptide chains are cross-linked by the transpeptidase (TP) (3, 29) (Fig. ​(Fig.1).1). The disaccharide unit N-acetylmuramyl(pentapeptide)-N-acetylglucosamine is assembled on a lipid carrier, undecaprenol phosphate, from the sugar precursors UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl(pentapeptide) [UDP-MurNAc(pp)], forming lipid II. The TG catalyzes the transfer of the disaccharide from lipid II to nascent peptidoglycan, releasing lipid pyrophosphate. FIG. 1. Schematic of stages 2 and 3 of peptidoglycan biosynthesis pathway indicating reference inhibitors of the enzymes. Cross-linked peptidoglycan is formed in wild-type membranes (A), but lipid II is formed in membranes of the E. coli AMA1004 ponB::Spcr strain ... Class A high-molecular-weight penicillin-binding proteins (PBPs), such as PBP1a and -1b of Escherichia coli, are bifunctional molecules having both transglycosylase and transpeptidase activity on the same polypeptide (20, 29, 31). Either PBP1a or PBP1b needs to be functional for the cell to survive; the double mutant is not viable (38). Since the PBPs are located on the periplasmic surface, they are more accessible to drug molecules; this location also precludes problems associated with the permeability of the cell wall to the drug and resistance due to drug efflux. Both transglycosylase and transpeptidase are attractive targets for drug discovery (14). However, both of these enzymes are difficult to assay in a format amenable to high-throughput screening; this is true for the transpeptidase in particular. The transglycosylase substrate, lipid II, is present in bacterial cells in small quantities and is tedious to isolate (21, 32). An alternative to isolating the substrate is allowing lipid II to accumulate in situ in membranes by using detergent to inhibit transglycosylation and subsequently removing the detergent to assay transglycosylation (5, 18). However, the product peptidoglycan is typically separated from lipid II by paper chromatography (5, 10). Using this method, the TG activity of PBP1b has been demonstrated previously (10, 15, 21, 29a, 30, 31). However, the activity of PBP1a (15) has been more difficult to measure, and the reaction may have to be performed on filter paper. More recently, lipid II and analogues have been chemically synthesized; these can be used to measure the TG activity, although the water solubility of the substrate is an issue and shorter-chain analogues are better substrates than the natural lipid (26, 33, 37). TG activity is inhibited by moenomycin (14, 31), which does not affect TP activity, and competition of its binding to the PBP has been proposed as an assay to isolate TG inhibitors (34). There are few reports of transpeptidase enzyme activity in membrane fractions (21, 31), since cross-linked peptidoglycan has very similar properties and cannot be separated from un-cross-linked precursor. The only means of measuring transpeptidation is by analysis of the degree of cross-linking of the peptidoglycan. This is a laborious procedure and needs large quantities of material and hence has more frequently been reported for whole cells (21, 31). The other method of measuring transpeptidase is by measuring the release of the terminal d-Ala from muramyl(pentapeptide) resulting from the cross-linking, which can be distinguished from carboxypeptidase by its dependence on peptidoglycan synthesis, i.e., inclusion of UDP-N-acetylglucosamine in the reaction (16). This method requires labeling of the terminal d-Ala residue, which is laborious, and measurement of d-Ala is not easily performed in a microplate. The hallmark of peptidoglycan transpeptidases is their inhibition by penicillin and related β-lactams, giving rise to the term “PBPs”; penicillin binds covalently to the PBPs and inhibits the TP, while having no effect on transglycosylation (12, 28). This has given rise to indirect assays as indicators of the TP activity of the PBPs, most of which are binding assays and are not truly reflective of the enzyme activity. The most commonly used assays involve the binding of a β-lactam to the PBPs to screen for agents that compete with this binding, the conversion of nitrocefin to a colored product upon binding to the PBP, or the use of a synthetic peptide that gives a colored product on hydrolysis (1, 2, 17, 25). These indirect assays can be easily adapted to microplates. An earlier report from our unit has shown that cross-linked peptidoglycan can be specifically detected in a microplate by capture with wheat germ agglutinin-coated scintillation proximity assay beads with detergent (8). In addition, this activity was shown to be a true measure of the enzyme activity of the TP (7). This makes it possible, in principle, to assay the coupled TG-TP activity, since cross-linked peptidoglycan can be distinguished from lipid II in a microplate format. Here we show that the plasmid-borne PBP1a gene can be transformed into an E. coli strain that lacks PBP1b, and its product can then be used to assay TG and TP activity. In vitro peptidoglycan synthesis, which is insignificant in the host strain, is reconstituted by overexpression of PBP1a. Further, the coupled peptidoglycan transglycosylase-transpeptidase of the PBPs can be directly measured in a two-step reaction. The substrate, lipid II, was made by incubating membranes of an E. coli strain lacking PBP1b with the peptidoglycan sugar precursors. In a second step, a source of PBP1b was added to these membranes to produce cross-linked peptidoglycan, which was captured using the scintillation proximity assay (SPA) beads. The assay can be used to screen in high throughput mode for inhibitors of both TG and TP. (Part of this work was presented previously in poster F-1545 by V. Ramachandran et al. at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 2004 [23].)

Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC 50 s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other β-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC 50 s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.

Microaerophilic adaptation has been described as one of the in vitro dormancy models for tuberculosis. Studies on Mycobacterium tuberculosis adapted to low oxygen levels showed an enhancement of glycine dehydrogenase (deaminating) activity. We studied the physiology of the fast-growing, nonpathogenic strain of Mycobacterium smegmatis ATCC 607 under low oxygen by shifting the actively growing M. smegmatis cells to static microaerophilic growth conditions. This shifting of M. smegmatis culture resulted in a similar phenomenon as seen with M. tuberculosis, i.e., elevated glycine dehydrogenase activity. Further purification of glycine dehydrogenase from M. smegmatis demonstrated glyoxylate amination, but failed to demonstrate glycine deamination, even in the purified fraction. Moreover, the purified protein showed pyruvate amination as well as L-alanine deamination activities. By activity staining, the protein band positive for glyoxylate amination demonstrated only pyruvate amination in the presence of NAD. Absence of glycine deamination activity strongly suggested that alanine dehydrogenase of M. smegmatis was responsible for glyoxylate amination in the cell lysate. This was further confirmed by demonstrating the similar level of upregulation of both glyoxylate and pyruvate amination activities in the cell lysate of the adapted culture.Key words: Mycobacterium smegmatis, glycine dehydrogenase, microaerophilic adaptation, alanine dehydrogenase.