RETRACTED ARTICLE: Antituberculars which target decaprenylphosphoryl-β-D-ribofuranose 2′-oxidase DprE1: state of art

Multidrug resistance is a major barrier in the battle against tuberculosis and still a leading cause of death worldwide. In order to fight this pathogen, two routes are practicable: vaccination or drug treatment. Vaccination against Mycobacterium tuberculosis with the current vaccine Mycobacterium bovis Bacillus Calmette–Guerin is partially successful, being its efficacy variable. A few new tuberculosis vaccines are now in various phases of clinical trials. The emergence of multidrug-resistant strains of M. tuberculosis gave the impulse to discover new effective antitubercular drugs, a few of which are in clinical development. Here we focus on three different classes of very promising antitubercular drugs recently discovered (benzothiazinones, dinitrobenzamides, and benzoquinoxalines) that share the same cellular target: a subunit of the heteromeric decaprenylphosphoryl-β-d-ribose 2′-epimerase, encoded by the dprE1 (or Rv3790) gene. This enzyme is involved in the biosynthesis of d-arabinose which is crucial for the synthesis of the mycobacterial cell wall and essential for the pathogen’s survival.     

Molecular modelling and comparative structural account of aspartyl β-semialdehyde dehydrogenase of Mycobacterium tuberculosis (H37Rv)

Aspartyl β-semialdehyde dehydrogenase (ASADH) is an important enzyme, occupying the first branch position of the biosynthetic pathway of the aspartate family of amino acids in bacteria, fungi and higher plants. It catalyses reversible dephosphorylation of l-β-aspartyl phosphate (βAP) to l-aspartate-β-semialdehyde (ASA), a key intermediate in the biosynthesis of diaminopimelic acid (DAP)—an essential component of cross linkages in bacterial cell walls. Since the aspartate pathway is unique to plants and bacteria, and ASADH is the key enzyme in this pathway, it becomes an attractive target for antimicrobial agent development. Therefore, with the objective of deducing comparative structural models, we have described a molecular model emphasizing the uniqueness of ASADH from Mycobacterium tuberculosis (H37Rv) that should generate insights into the structural distinctiveness of this protein as compared to structurally resolved ASADH from other bacterial species. We find that mtASADH exhibits structural features common to bacterial ASADH, while other structural motifs are not present. Structural analysis of various domains in mtASADH reveals structural conservation among all bacterial ASADH proteins. The results suggest that the probable mechanism of action of the mtASADH enzyme might be same as that of other bacterial ASADH. Analysis of the structure of mtASADH will shed light on its mechanism of action and may help in designing suitable antagonists against this enzyme that could control the growth of Mycobacterium tuberculosis. Anupama Singh and Hemant R. Kushwaha contributed equally to this work.     

Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme

The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 Å resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity.     

Structural analyses of a purine biosynthetic enzyme from Mycobacterium tuberculosis reveal a novel bound nucleotide

Enzymes of the de novo purine biosynthetic pathway have been identified as essential for the growth and survival of Mycobacterium tuberculosis and thus have potential for the development of anti-tuberculosis drugs. The final two steps of this pathway are carried out by the bifunctional enzyme 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC), also known as PurH. This enzyme has already been the target of anti-cancer drug development. We have determined the crystal structures of the M. tuberculosis ATIC (Rv0957) both with and without the substrate 5-aminoimidazole-4-carboxamide ribonucleotide, at resolutions of 2.5 and 2.2 Å, respectively. As for other ATIC enzymes, the protein is folded into two domains, the N-terminal domain (residues 1–212) containing the cyclohydrolase active site and the C-terminal domain (residues 222–523) containing the formyltransferase active site. An adventitiously bound nucleotide was found in the cyclohydrolase active site in both structures and was identified by NMR and mass spectral analysis as a novel 5-formyl derivative of an earlier intermediate in the biosynthetic pathway 4-carboxy-5-aminoimidazole ribonucleotide. This result and other studies suggest that this novel nucleotide is a cyclohydrolase inhibitor. The dimer formed by M. tuberculosis ATIC is different from those seen for human and avian ATICs, but it has a similar ∼50-Å separation of the two active sites of the bifunctional enzyme. Evidence in M. tuberculosis ATIC for reactivity of half-the-sites in the cyclohydrolase domains can be attributed to ligand-induced movements that propagate across the dimer interface and may be a common feature of ATIC enzymes.     

An in silico structural insights into Plasmodium LytB protein and its inhibition

In most of the pathogenic organisms including Plasmodium falciparum, isoprenoids are synthesized via MEP (MethylErythritol 4-Phosphate) pathway. LytB is the last enzyme of this pathway which catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Since the MEP pathway is not used by humans, it represents an attractive target for the development of new anti-malarial compounds or inhibitors. Here a systematic in silico study has been conducted to get an insight into the structure of Plasmodium lytB as well as its affinities towards different inhibitors. We used comparative modeling technique to predict the three-dimensional (3D) structure of Plasmodium LytB taking Escherichia coli LytB protein (PDB ID: 3KE8) as template and the model was subsequently refined through molecular dynamics (MD) simulation. A large ligand data-set containing diphospate group was subjected for virtual screening against the target using GOLD 5.2 program. Considering the mode of binding and affinities, 17 leads were selected on basis of binding energies in comparison to its substrate HMBPP (Gold.Chemscore.DG: -20.9734 kcal/mol). Among them, five were discarded because of their inhibitory activity towards other human enzymes. The rest 12 potential leads carry all the properties of any “drug like” molecule and the knowledge of Plasmodium LytB-inhibitory mechanism which can provide valuable support for the anti-malarial-inhibitor design in future.     

The structure of glucose‐1‐phosphate thymidylyltransferase from Mycobacterium tuberculosis reveals the location of an essential magnesium ion in the RmlA‐type enzymes

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, continues to be a major threat to populations worldwide. Whereas the disease is treatable, the drug regimen is arduous at best with the use of four antimicrobials over a six‐month period. There is clearly a pressing need for the development of new therapeutics. One potential target for structure‐based drug design is the enzyme RmlA, a glucose‐1‐phosphate thymidylyltransferase. This enzyme catalyzes the first step in the biosynthesis of l ‐rhamnose, which is a deoxysugar critical for the integrity of the bacterium's cell wall. Here, we report the X‐ray structures of M. tuberculosis RmlA in complex with either dTTP or dTDP‐glucose to 1.6 Å and 1.85 Å resolution, respectively. In the RmlA/dTTP complex, two magnesium ions were observed binding to the nucleotide, both ligated in octahedral coordination spheres. In the RmlA/dTDP‐glucose complex, only a single magnesium ion was observed. Importantly, for RmlA‐type enzymes with known three‐dimensional structures, not one model shows the position of the magnesium ion bound to the nucleotide‐linked sugar. As such, this investigation represents the first direct observation of the manner in which a magnesium ion is coordinated to the RmlA product and thus has important ramifications for structure‐based drug design. In the past, molecular modeling procedures have been employed to derive a three‐dimensional model of the M. tuberculosis RmlA for drug design. The X‐ray structures presented herein provide a superior molecular scaffold for such endeavors in the treatment of one of the world's deadliest diseases.     

Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of K_M ^DXP = 252 µM and K_M ^NADPH = 13 µM, IC₅₀ values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and K_i values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.     

Catharanthus terpenoid indole alkaloids: biosynthesis and regulation

Catharanthus roseus is still the only source for the powerful antitumour drugs vinblastine and vincristine. Some other pharmaceutical compounds from this plant, ajmalicine and serpentine are also of economical importance. Although C. roseus has been studied extensively and was subject of numerous publications, a full characterization of its alkaloid pathway is not yet achieved. Here we review some of the recent work done on this plant. Most of the work focussed on early steps of the pathway, particularly the discovery of the 2-C-methyl-d-erythritol 4-phosphate (MEP)-pathway leading to terpenoids. Both mevalonate and MEP pathways are utilized by plants with apparent cross-talk between them across different compartments. Many genes of the early steps in Catharanthus alkaloid pathway have been cloned and overexpressed to improve the biosynthesis. Research on the late steps in the pathway resulted in cloning of several genes. Enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their localization and regulation are discussed. Much progress has been made at alkaloid regulatory level. Feeding precursors, growth regulators treatments and metabolic engineering are good tools to increase productivity of terpenoid indole alkaloids. But still our knowledge of the late steps in the Catharanthus alkaloid pathway and the genes involved is limited.     

Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug‐resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug‐resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug‐resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A‐site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem‐loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug‐resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug‐resistant tuberculosis disease.     

Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23)

The emergence of drug-resistant strains of Mycobacterium tuberculosis, the major causative agent of tuberculosis (TB), and the deadly HIV-TB co-infection have led to an urgent need for the development of new anti-TB drugs. The histidine biosynthetic pathway is present in bacteria, archaebacteria, lower eukaryotes and plants, but is absent in mammals. Disruption of the hisD gene has been shown to be essential for M. tuberculosis survival. Here we present cloning, expression and purification of recombinant hisD-encoded histidinol dehydrogenase (MtHisD). N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtHisD. Analytical gel filtration, metal requirement analysis, steady-state kinetics and isothermal titration calorimetry data showed that homodimeric MtHisD is a metalloprotein that follows a Bi Uni Uni Bi Ping-Pong mechanism. pH-rate profiles and a three-dimensional model of MtHisD allowed proposal of amino acid residues involved in either catalysis or substrate(s) binding.     

The two 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition

Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 μM for phosphoenolpyruvate and 500 μM for erythrose 4-phosphate. The rate constant was calculated as 6 s^–1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (K _i of 0.9 μM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate. Received: 29 December 1997 / Accepted: 3 March 1998     

Molecular modelling of Mycobacterium tuberculosis acetolactate synthase catalytic subunit and its molecular docking study with inhibitors

Mycobacterium tuberculosis is a leading cause of infectious disease in the world today. This outlook is aggravated by a growing number of M. tuberculosis infections in individuals who are immunocompromised as a result of HIV infections. Thus, new and more potent anti-TB agents are necessary. Therefore, acetolactate synthase (mtALS) was selected as a target enzyme to combat M. tuberculosis. In this work, the three-dimensional molecular model of the hypothetical structure for the ALS catalytic subunit of M. tuberculosis was elucidated by homology modelling. In addition, the orientations and binding affinities of sulfonylurea inhibitors with the new structure was investigated. Our findings could be helpful for the design of new, more potent mtAHAS inhibitors.     

New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis

Background

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.

Methodology/Principal Findings

To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the ‘hits’ for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.

Conclusions/Significance

This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.     

Identification and validation of l-asparaginase as a potential metabolic target against Mycobacterium tuberculosis

Multidrug-resistant Mycobacterium tuberculosis (Mtb) has emerged as a major health challenge, necessitating the search for new molecular targets. A secretory amidohydrolase, l -asparaginase of Mtb (MtA), originally implicated in nitrogen assimilation and neutralization of acidic microenvironment inside human alveolar macrophages, has been proposed as a crucial metabolic enzyme. To investigate whether this enzyme could serve as a potential drug target, it was studied for structural details and active site–specific inhibitors were tested on cultured Mycobacterial strain. The structural details of MtA obtained through comparative modeling and molecular dynamics simulations provided insights about the orchestration of an alternate reaction mechanism at the active site. This was contrary to the critical Tyr flipping mechanism reported in other asparaginases. We report the novel finding of Tyr to Val replacement in catalytic triad I along with the structural reorganization of a β-hairpin loop upon substrate binding in MtA active site. Further, 5 MtA-specific, active-site–based inhibitors were obtained by following a rigorous differential screening protocol. When tested on Mycobacterium culture, 3 of these, M3 (ZINC 4740895), M26 (ZINC 33535), and doxorubicin showed promising results with inhibitory concentrations (IC ₅₀) of 431, 100, and 56 µM, respectively. Based on our findings and considering stark differences with human asparaginase, we project MtA as a promising molecular target against which the selected inhibitors may be used to counteract Mtb infection effectively.     

Insights from the docking and molecular dynamics simulation of the Phosphopantetheinyl transferase (PptT) structural model from Mycobacterium tuberculosis

A great challenge is posed to the treatment of tuberculosis due to the evolution of multidrug-resistant (MDR) and extensively drugresistant (XDR) strains of Mycobacterium tuberculosis in recent times. The complex cell envelope of the bacterium contains unusual structures of lipids which protects the bacterium from host enzymes and escape immune response. To overcome the drug resistance, targeting “drug targets” which have a critical role in growth and virulence factor is a novel approach for better tuberculosis treatment. The enzyme Phosphopantetheinyl transferase (PptT) is an attractive drug target as it is primarily involved in post translational modification of various types-I polyketide synthases and assembly of mycobactin, which is required for lipid virulence factors. Our in silico studies reported that the structural model of M.tuberculosis PptT characterizes the structure-function activity. The refinement of the model was carried out with molecular dynamics simulations and was analyzed with root mean square deviation (RMSD), and radius of gyration (Rg). This confirmed the structural behavior of PptT in dynamic system. Molecular docking with substrate coenzyme A (CoA) identified the binding pocket and key residues His93, Asp114 and Arg169 involved in PptT-CoA binding. In conclusion, our results show that the M.tuberculosis PptT model and critical CoA binding pocket initiate the inhibitor design of PptT towards tuberculosis treatment.     

Isolation and characterization of a d-glucose/xylose isomerase from a new thermophilic strain Streptomyces sp. (PLC)

A thermostable d-xylase isomerase from a newly isolated thermophilic Streptomyces sp. (PLC) strain is described. The enzyme was purified to homogeneity. It is a homotetramer with a native molecular mass of 183 kDa and a subunit molecular mass of 46 kDa. The enzyme has a K _m of 35 mM for d-xylose and also accepts d-glucose as substrate, however, with a tenfold higher K _m (0.4 M) and half the maximum velocity. Both the activity and stability of this d-xylose isomerase depend strongly on divalent metal ions. Two metal ions bind per subunit to non-identical sites. Mg²⁺, Mn²⁺ and Co²⁺ are of comparable efficiency for the d-xylose isomerase reaction. Con²⁺ is the most efficient cofactor for d-glucose isomerization. The enzyme remains fully active up to 95°C. The activity decreases at 53°C in the presence of Co²⁺ and Mg²⁺ with a half-life of 7 and 9 days respectively. In the presence of Mn²⁺ the enzyme activity remains constant for at least 10 days and at 70°C 50% of the activity is lost after 5 days.     

Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K_M^DXP = 104 µM, K_M^NADPH = 13 µM, k_cat^DXP = 2 s⁻¹, k_cat^NADPH = 1.3 s⁻¹), an IC₅₀ for fosmidomycin of 247 nM, and a K_i for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg⁺² as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.     

Active site conformational changes upon reaction intermediate biotinyl‐5'‐AMP binding in biotin protein ligase from Mycobacterium tuberculosis

Protein biotinylation, a rare form of post‐translational modification, is found in enzymes required for lipid biosynthesis. In mycobacteria, this process is essential for the formation of their complex and distinct cell wall and has become a focal point of drug discovery approaches. The enzyme responsible for this process, biotin protein ligase, substantially varies in different species in terms of overall structural organization, regulation of function and substrate specificity. To advance the understanding of the molecular mechanism of biotinylation in Mycobacterium tuberculosis we have biochemically and structurally characterized the corresponding enzyme. We report the high‐resolution crystal structures of the apo‐form and reaction intermediate biotinyl‐5'‐AMP‐bound form of M. tuberculosis biotin protein ligase. Binding of the reaction intermediate leads to clear disorder‐to‐order transitions. We show that a conserved lysine, Lys138, in the active site is essential for biotinylation.     

Hypoxanthine–guanine phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv: Cloning, expression, and biochemical characterization

Human tuberculosis (TB) is a major cause of morbidity and mortality worldwide, especially in poor and developing countries. Moreover, the emergence of Mycobacterium tuberculosis strains resistant to first- and second-line anti-TB drugs raises the prospect of virtually incurable TB. Enzymes of the purine phosphoribosyltransferase (PRTase) family are components of purine salvage pathway and have been proposed as drug targets for the development of chemotherapeutic agents against infective and parasitic diseases. The PRTase-catalyzed chemical reaction involves the ribophosphorylation in one step of purine bases (adenine, guanine, hypoxanthine, or xanthine) and their analogues to the respective nucleoside 5′-monophosphate and pyrophosphate. Hypoxanthine–guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) is a purine salvage pathway enzyme that specifically recycles hypoxanthine and guanine from the medium, which are in turn converted to, respectively, IMP and GMP. Here we report cloning, DNA sequencing, expression in Escherichia coli BL21 (DE3) cells, purification to homogeneity, N-terminal amino acid sequencing, mass spectrometry analysis, and determination of apparent steady-state kinetic parameters for an in silico predicted M. tuberculosis HGPRT enzyme. These data represent an initial step towards future functional and structural studies, and provide a solid foundation on which to base M. tuberculosis HGPRT-encoding gene manipulation experiments to demonstrate its role in the biology of the bacillus.     

Deciphering kas operon locus in Mycobacterium aurum and genesis of a recombinant strain for rational-based drug screening

Aims: To generate a recombinant Mycobacterium aurum strain for screening of antimycobacterial compounds affecting fatty acid synthase type II (FAS-II) elongation pathway. Methods and Results: kas operon locus was delineated in M. aurum, a fast growing nonpathogenic strain. Cloning and sequencing all the genes of the operon showed similar organization and sequence similarities with Mycobacterium tuberculosis (H37Rv) orthologues. Further, we cloned the upstream region of M. tuberculosis kas operon in fusion with lacZ reporter gene and put it in M. aurum. Recombinant M. aurum strain showed continued expression of reporter gene throughout the growth while an increased expression of the reporter gene was noticed only after treatment with FAS-II pathway inhibitors. Swapping of the regulatory sequence aborts the increased reporter gene expression after same antibiotic treatments. Conclusions: kas operon genes are similarly organized in M. tuberculosis and M. aurum. H37Rv kas operon promoter upregulates the reporter gene expression in M. aurum only upon treatment with drugs inhibiting FAS-II pathway. Significance and Impact of the Study: It would serve as a good second-line screen for characterization of compounds showing antimycobacterial activity in a first-line screen. With the simplicity of β-galactosidase enzyme assay the system can be easily adapted in high-throughput mode.