Kinetic and chemical mechanisms of shikimate dehydrogenase from Mycobacterium tuberculosis

Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) catalyzes the fourth reaction in the shikimate pathway, the NADPH-dependent reduction of 3-dehydroshikimate. To gather information on the kinetic mechanism, initial velocity patterns, product inhibition, and primary deuterium kinetic isotope effect studies were performed and the results suggested a steady-state ordered bi-bi kinetic mechanism. The magnitudes of both primary and solvent kinetic isotope effects indicated that the hydride transferred from NADPH and protons transferred from the solvent in the catalytic cycle are not significantly rate limiting in the overall reaction. Proton inventory analysis indicates that one proton gives rise to solvent isotope effects. Multiple isotope effect studies indicate that both hydride and proton transfers are concerted. The pH profiles revealed that acid/base chemistry takes place in catalysis and substrate binding. The MtbSD 3D model was obtained in silico by homology modeling. Kinetic and chemical mechanisms for MtbSD are proposed on the basis of experimental data.     

Expression, purification and properties of shikimate dehydrogenase from Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the main diseases to mankind. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. The shikimate pathway is considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammalian cells. The Mycobacterium tuberculosis aroE-encoded shikimate dehydrogenase was cloned, expressed and purified. Sequence alignment analysis shows that shikimate dehydrogenase of Mycobacterium tuberculosis exhibit the pattern of G-X-(N/S)-V-(T/S)-X-PX-K, which is highly conserved within the shikimate dehydrogenase family. The recombinant shikimate dehydrogenase spectrum determined by CD spectroscopy showed that the percentages for alpha-helix, beta-sheet, beta-turn, and random coil were 29.2 %, 9.3 %, 32.7 %, and 28.8 %, respectively. The enzymatic characterization demonstrates that it appears to be fully active at pH from 9.0 to 12, and temperature 63(o)C. The apparent Michaelis constant for shikimic acid and NADP(+) were calculated to be about 29.5 microM and 63 microM. The recombinant shikimate dehydrogenase catalyzes the substrate in the presence of NADP(+) with an enzyme turnover number of 399 s(-1). Zymological studies suggest that the cloned shikimate dehydrogenase from M. tuberculosis has a pretty activity, and the work should help in the discovery of enzyme inhibitors and further of possible antimicrobial agents against Mycobacterium tuberculosis.     

Functional shikimate dehydrogenase from Mycobacterium tuberculosis H37Rv: purification and characterization

Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded shikimate dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis shikimate dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents.     

Potent inhibitors of a shikimate pathway enzyme from Mycobacterium tuberculosis: combining mechanism- and modeling-based design

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.     

Cloning and expression of functional shikimate dehydrogenase (EC 1.1.1.25) from Mycobacterium tuberculosis H37Rv

tau-Crystallin is a taxon-restricted crystallin found in eye lenses of reptiles and a few avian species but presumably absent in mammals. The level of tau-crystallin in the lens varies among different species. In the crocodile lens, it is the least abundant crystallin and is present in trace amounts. We present a method for cloning, overexpression, and purification of crocodilian tau-crystallin utilizing a combination of gel filtration and ion-exchange chromatography yielding an extremely purified protein. The protein gets profusely expressed resulting in a fairly high yield and exists as a monomeric entity of 47.5 kDa molecular mass. The recombinant tau-crystallin exists in a properly folded native state as probed by circular dichroism and fluorescence spectroscopy and exhibits enolase activity.     

Validation of the essential ClpP protease in Mycobacterium tuberculosis as a novel drug target

Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.     

Understanding the transmission of Mycobacterium ulcerans: A step towards controlling Buruli ulcer

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rare but chronic debilitating skin and soft tissue disease found predominantly in West Africa and Southeast Australia. While a moderate body of research has examined the distribution of M. ulcerans, the specific route(s) of transmission of this bacterium remain unknown, hindering control efforts. M. ulcerans is considered an environmental pathogen given it is associated with lentic ecosystems and human-to-human spread is negligible. However, the pathogen is also carried by various mammals and invertebrates, which may serve as key reservoirs and mechanical vectors, respectively. Here, we examine and review recent evidence from these endemic regions on potential transmission pathways, noting differences in findings between Africa and Australia, and summarising the risk and protective factors associated with Buruli ulcer transmission. We also discuss evidence suggesting that environmental disturbance and human population changes precede outbreaks. We note five key research priorities, including adoption of One Health frameworks, to resolve transmission pathways and inform control strategies to reduce the spread of Buruli ulcer.     

Proteins with complex architecture as potential targets for drug design: a case study of Mycobacterium tuberculosis

Lengthy co-evolution of Homo sapiens and Mycobacterium tuberculosis, the main causative agent of tuberculosis, resulted in a dramatically successful pathogen species that presents considerable challenge for modern medicine. The continuous and ever increasing appearance of multi-drug resistant mycobacteria necessitates the identification of novel drug targets and drugs with new mechanisms of action. However, further insights are needed to establish automated protocols for target selection based on the available complete genome sequences. In the present study, we perform complete proteome level comparisons between M. tuberculosis, mycobacteria, other prokaryotes and available eukaryotes based on protein domains, local sequence similarities and protein disorder. We show that the enrichment of certain domains in the genome can indicate an important function specific to M. tuberculosis. We identified two families, termed pkn and PE/PPE that stand out in this respect. The common property of these two protein families is a complex domain organization that combines species-specific regions, commonly occurring domains and disordered segments. Besides highlighting promising novel drug target candidates in M. tuberculosis, the presented analysis can also be viewed as a general protocol to identify proteins involved in species-specific functions in a given organism. We conclude that target selection protocols should be extended to include proteins with complex domain architectures instead of focusing on sequentially unique and essential proteins only.     

Kinetic mechanism determination and analysis of metal requirement of dehydroquinate synthase from Mycobacterium tuberculosis H37Rv: an essential step in the function-based rational design of anti-TB drugs

The number of new cases of tuberculosis (TB) arising each year is increasing globally. Migration, socio-economic deprivation, HIV co-infection and the emergence of drug-resistant strains of Mycobacterium tuberculosis, the main causative agent of TB in humans, have all contributed to the increasing number of TB cases worldwide. Proteins that are essential to the pathogen survival and absent in the host, such as enzymes of the shikimate pathway, are attractive targets to the development of new anti-TB drugs. Here we describe the metal requirement and kinetic mechanism determination of M. tuberculosis dehydroquinate synthase (MtDHQS). True steady-state kinetic parameters determination and ligand binding data suggested that the MtDHQS-catalyzed chemical reaction follows a rapid-equilibrium random mechanism. Treatment with EDTA abolished completely the activity of MtDHQS, and addition of Co(2+) and Zn(2+) led to, respectively, full and partial recovery of the enzyme activity. Excess Zn(2+) inhibited the MtDHQS activity, and isotitration microcalorimetry data revealed two sequential binding sites, which is consistent with the existence of a secondary inhibitory site. We also report measurements of metal concentrations by inductively coupled plasma atomic emission spectrometry. The constants of the cyclic reduction and oxidation of NAD(+) and NADH, respectively, during the reaction of MtDHQS was monitored by a stopped-flow instrument, under single-turnover experimental conditions. These results provide a better understanding of the mode of action of MtDHQS that should be useful to guide the rational (function-based) design of inhibitors of this enzyme that can be further evaluated as anti-TB drugs.     

Prioritizing genomic drug targets in pathogens: application to Mycobacterium tuberculosis

We have developed a software program that weights and integrates specific properties on the genes in a pathogen so that they may be ranked as drug targets. We applied this software to produce three prioritized drug target lists for Mycobacterium tuberculosis, the causative agent of tuberculosis, a disease for which a new drug is desperately needed. Each list is based on an individual criterion. The first list prioritizes metabolic drug targets by the uniqueness of their roles in the M. tuberculosis metabolome ("metabolic chokepoints") and their similarity to known "druggable" protein classes (i.e., classes whose activity has previously been shown to be modulated by binding a small molecule). The second list prioritizes targets that would specifically impair M. tuberculosis, by weighting heavily those that are closely conserved within the Actinobacteria class but lack close homology to the host and gut flora. M. tuberculosis can survive asymptomatically in its host for many years by adapting to a dormant state referred to as "persistence." The final list aims to prioritize potential targets involved in maintaining persistence in M. tuberculosis. The rankings of current, candidate, and proposed drug targets are highlighted with respect to these lists. Some features were found to be more accurate than others in prioritizing studied targets. It can also be shown that targets can be prioritized by using evolutionary programming to optimize the weights of each desired property. We demonstrate this approach in prioritizing persistence targets.     

The crystal structure of Mycobacterium tuberculosis alkylhydroperoxidase AhpD, a potential target for antitubercular drug design

The resistance of Mycobacterium tuberculosis to isoniazid is commonly linked to inactivation of a catalase-peroxidase, KatG, that converts isoniazid to its biologically active form. Loss of KatG is associated with elevated expression of the alkylhydroperoxidases AhpC and AhpD. AhpD has no sequence identity with AhpC or other proteins but has alkylhydroperoxidase activity and possibly additional physiological activities. The alkylhydroperoxidase activity, in the absence of KatG, provides an important antioxidant defense. We have determined the M. tuberculosis AhpD structure to a resolution of 1.9 A. The protein is a trimer in a symmetrical cloverleaf arrangement. Each subunit exhibits a new all-helical protein fold in which the two catalytic sulfhydryl groups, Cys-130 and Cys-133, are located near a central cavity in the trimer. The structure supports a mechanism for the alkylhydroperoxidase activity in which Cys-133 is deprotonated by a distant glutamic acid via the relay action of His-137 and a water molecule. The cysteine then reacts with the peroxide to give a sulfenic acid that subsequently forms a disulfide bond with Cys-130. The crystal structure of AhpD identifies a new protein fold relevant to members of this protein family in other organisms. The structural details constitute a potential platform for the design of inhibitors of potential utility as antitubercular agents and suggest that AhpD may have disulfide exchange properties of importance in other areas of M. tuberculosis biology.     

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.     

TB or Not TB: how Mycobacterium tuberculosis may evade drug treatment

In this issue of Cell, a study by Valerie Mizrahi and her colleagues suggests that a putative error-prone DNA polymerase encoded by the dnaE2 gene of Mycobacterium tuberculosis may bypass certain types of DNA base damage, generating mutations. This may be an important mechanism for generating drug-resistant strains of M. tuberculosis.     

Design and optimization of a recombinant system for large-scale production of the MPT64 antigen from Mycobacterium tuberculosis

Early clinical trials of a potential new tuberculosis (TB) diagnostic, the Patch Test for Active TB (PTAT), used MPB64 protein that was purified from the spent medium of Bacillus Calmette-Guérin (BCG) Tokyo 172 vaccine production. The yield was poor, 0.05 mg/L, and the process for purification of the protein was complex, requiring four chromatographic steps. The combination of yield and purification complexity compromised the ability to produce the PTAT diagnostic in quantities sufficient for larger clinical trials and commercialization. We report here a highly efficient method for the overexpression and purification of recombinant MPT64 from Escherichia coli (rMPT64) based upon a mild insolubility of rMPT64 following induction, and scalable anion-exchange and gel filtration chromatographies. Yields of protein were improved substantially to approximately 250 mg/L, and resulted in a preparation greater than 98% pure. Quantitative release assays were developed and used with MALDI-TOF mass spectrometry to confirm the identity of rMPT64. Using a guinea pig model of active TB, we found that rMPT64 elicited a specific immune response indistinguishable from that of MPB64 purified from BCG Tokyo culture filtrates. These results describe the first efficient and scalable protocol for production of rMPT64, demonstrate its activity in an animal model of active TB, and lay the foundation of ongoing and future use of the PTAT in clinical settings.     

Mycobacterium tuberculosis cytochrome P450 enzymes: a cohort of novel TB drug targets

TB (tuberculosis) disease remains responsible for the death of over 1.5 million people each year. The alarming emergence of drug-resistant TB has sparked a critical need for new front-line TB drugs with a novel mode of action. In the present paper, we review recent genomic and biochemical evidence implicating Mycobacterium tuberculosis CYP (cytochrome P450) enzymes as exciting potential targets for new classes of anti-tuberculars. We also discuss HTS (high-throughput screening) and fragment-based drug-discovery campaigns that are being used to probe their potential druggability.