The functions of OmpATb,a pore-forming protein of Mycobacterium tuberculosis

The functions of OmpATb, the product of the ompATb gene of Mycobacterium tuberculosis and a putative porin, were investigated by studying a mutant with a targeted deletion of the gene, and by observing expression of the gene in wild-type M. tuberculosis H37Rv by real-time polymerase chain reaction (PCR) and immunoblotting. The loss of ompATb had no effect on growth under normal conditions, but caused a major reduction in ability to grow at reduced pH. The gene was substantially upregulated in wild-type bacteria exposed to these conditions. The mutant was impaired in its ability to grow in macrophages and in normal mice, although it was as virulent as the wild type in mice that lack T cells. Deletion of the ompATb gene reduced permeability to several small water-soluble substances. This was particularly evident at pH 5.5; at this pH, uptake of serine was minimal, suggesting that, at this pH, OmpATb might be the only functioning porin. These data indicate that OmpATb has two functions: as a pore-forming protein with properties of a porin, and in enabling M. tuberculosis to respond to reduced environmental pH. It is not known whether this second function is related to the porin-like activity at low pH or involves a completely separate role for OmpATB. The involvement with pH is likely to contribute to the ability of M. tuberculosis to overcome host defence mechanisms and grow in a mammalian host.     

Structural mechanics of the pH-dependent activity of beta-carbonic anhydrase from Mycobacterium tuberculosis

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. Carbonic anhydrases are divided into at least three classes and are believed to share a zinc-hydroxide mechanism for carbon dioxide hydration. beta-carbonic anhydrases are broadly spread among the domains of life, and existing structures from different organisms show two distinct active site setups, one with three protein coordinations to the zinc (accessible) and the other with four (blocked). The latter is believed to be inconsistent with the zinc-hydroxide mechanism. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a beta-carbonic anhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. We have recently solved the structure of this protein, which was a dimeric protein with a blocked active site. Here we present the structure of the thiocyanate complexed protein in a different crystal form. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a beta-carbonic anhydrase can switch between the two states. A pH-dependent dimer to tetramer equilibrium was also demonstrated by dynamic light scattering measurements. The data presented here, therefore, suggest a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium.     

Structure of the Mycobacterium tuberculosis OmpATb protein: a model of an oligomeric channel in the mycobacterial cell wall

The pore-forming outer membrane protein OmpATb from Mycobacterium tuberculosis is a virulence factor required for acid resistance in host phagosomes. In this study, we determined the 3D structure of OmpATb by NMR in solution. We found that OmpATb is composed of two independent domains separated by a proline-rich hinge region. As expected, the high-resolution structure of the C-terminal domain (OmpATb(198-326)) revealed a module structurally related to other OmpA-like proteins from Gram-negative bacteria. The N-terminal domain of OmpATb (73-204), which is sufficient to form channels in planar lipid bilayers, exhibits a fold, which belongs to the α+β sandwich class fold. Its peculiarity is to be composed of two overlapping subdomains linked via a BON (Bacterial OsmY and Nodulation) domain initially identified in bacterial proteins predicted to interact with phospholipids. Although OmpATb(73-204) is highly water soluble, current-voltage measurements demonstrate that it is able to form conducting pores in model membranes. A HADDOCK modeling of the NMR data gathered on the major monomeric form and on the minor oligomeric populations of OmpATb(73-204) suggest that OmpATb(73-204) can form oligomeric rings able to insert into phospholipid membrane, similar to related proteins from the Type III secretion systems, which form multisubunits membrane-associated rings at the basal body of the secretion machinery.     

The N-terminal domain of OmpATb is required for membrane translocation and pore-forming activity in mycobacteria

OmpATb is the prototype of a new family of porins in Mycobacterium tuberculosis and Mycobacterium bovis BCG. Although the pore-forming activity of this protein has been clearly established by using recombinant protein produced in Escherichia coli, characterization of the native porin has been hampered by the scarce amount of protein present in the M. tuberculosis detergent extracts. To this aim, we have developed a protocol to overproduce and obtain high yields of OmpATb in both Mycobacterium smegmatis and M. bovis BCG. The protein could be extracted and purified from the cell wall fraction and subsequently used for analysis of the pore-forming activity in multichannel and single-channel conductance experiments. Our results indicate that OmpATb produced in mycobacteria presents an average conductance value of 1,600+/-100 pS, slightly higher than that of OmpATb produced in E. coli, suggesting the occurrence of OmpATb in a highly ordered organization within the mycobacterial cell wall. In contrast to OmpATb, a truncated form lacking the first 72 amino acids (OmpATb73-326) was essentially found in the cytosol and was not active in planar lipid bilayers. This suggested that the N-terminal domain of OmpATb could participate in targeting of OmpATb to the cell wall. This was further confirmed by analyzing M. smegmatis clones expressing a chimeric protein consisting of a fusion between the N-terminal domain of OmpATb and the E. coli PhoA reporter. The present study shows for the first time that the N terminus of OmpATb is required for targeting the porin to the cell wall and also appears to be essential for its pore-forming activity.     

Purification and characterization of the acetyl-CoA synthetase from Mycobacterium tuberculosis

Acetyl-CoA (AcCoA) synthetase (Acs) catalyzes the conversion of acetate into AcCoA, which is involved in many catabolic and anabolic pathways. Although this enzyme has been studied for many years in many organisms, the properties of Mycobacterium tuberculosis Acs and the regulation of its activity remain unknown. Here, the putative acs gene of M. tuberculosis H37Rv (Mt-Acs) was expressed as a fusion protein with 6×His-tag on the C-terminus in Escherichia coli. The recombinant Mt-Acs protein was successfully purified and then its enzymatic characteristics were analyzed. The optimal pH and temperature, and the kinetic parameters of Mt-Acs were determined. To investigate whether Mt-Acs is regulated by lysine acetylation as reported for Salmonella enterica Acs, its mutant K617R was also generated. Determination of the enzymatic activity suggests that Lys-617 is critical for its function. We further demonstrated that Mt-Acs underwent auto-acetylation with acetate but not with AcCoA as the acetyl donor, which resulted in the decrease of its activity. CoA, the substrate for AcCoA formation, inhibited the auto-acetylation. Furthermore, the silent information regulator (Sir2) of M. tuberculosis (Mt-Sir2) could catalyze Mt-Acs deacetylation, which resulted in activation of Acs. These results may provide more insights into the physiological roles of Mt-Acs in M. tuberculosis central metabolism.     

Purification and characterization of beta-glucuronidase inhibitor from Mycobacterium tuberculosis

Factors inhibitory to beta-glucuronidase were found in the culture filtrate and in a bacillary extract of Mycobacterium tuberculosis H37Rv grown for 6 weeks on Sauton medium. The inhibitors were purified by ammonium sulfate fractionation, treatment with n-butanol and streptomycin, and chromatography on DEAE-Sepharose CL-6B. Two inhibitors were obtained from the culture filtrate. The molecular weights were estimated to be 25,500 and 15,500 by gel filtration on a Sephadex G-75 column. Three inhibitors were purified from the bacillary extract, two of which were similar to those from the culture filtrate. The molecular weight of the third inhibitor was 21,000. However, the molecular weight of all the denatured inhibitors was 8,600 in the presence of sodium dodecyl sulfate. The inhibitors contained extremely high amounts of glutamic and aspartic acids and had a highly acidic isoelectric point of pH 2.5. The inhibitors acted noncompetitively against beta-glucuronidase of guinea pig origin at an optimal pH 4.5. beta-Glucuronidases from human peripheral leukocytes and beef liver were partially sensitive to the inhibitors; all the other enzymes tested for sensitivity were unaffected by the inhibitors.     

Cloning and characterization of the aroA gene from Mycobacterium tuberculosis.

The aroA gene from Mycobacterium tuberculosis has been cloned by complementation of an aroA mutant of Escherichia coli after lysogenization with a recombinant DNA library in the lambda gt11 vector. Detailed characterization of the M. tuberculosis aroA gene by nucleotide sequencing and by immunochemical analysis of the expressed product indicates that it encodes a 5-enolpyruvylshikimate-3-phosphate synthase that is structurally related to analogous enzymes from other bacterial, fungal, and plant sources. The potential use of the cloned gene in construction of genetically defined mutant strains of M. tuberculosis by gene replacement is proposed as a novel approach to the rational attenuation of mycobacterial pathogens and the possible development of new antimycobacterial vaccines.     

Functional characterization of a novel ArgA from Mycobacterium tuberculosis

The Mycobacterium tuberculosis gene Rv2747 encodes a novel 19-kDa ArgA that catalyzes the initial step in L-arginine biosynthesis, namely the conversion of L-glutamate to alpha-N-acetyl-L-glutamate. Initial velocity studies reveal that Rv2747 proceeds through a sequential kinetic mechanism, with K(m) values of 280 mM for L-glutamine and 150 microM for acetyl-coenzyme A and with a k(cat) value of 200 min(-1). Initial velocity studies with L-glutamate showed that even at concentrations of 600 mM, saturation was not observed. Therefore, only a k(cat)/K(m) value of 125 M(-1) min(-1) can be calculated. Inhibition studies reveal that the enzyme is strongly regulated by L-arginine, the end product of the pathway (50% inhibitory concentration, 26 microM). The enzyme was completely inhibited by 500 microM arginine, with a Hill coefficient of 0.60, indicating negatively cooperative binding of L-arginine.     

Functional and structural characterization of a thiol peroxidase from Mycobacterium tuberculosis

A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H2O2, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 A shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Calpha atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.     

Identification and characterization of a unique adenosine kinase from Mycobacterium tuberculosis

Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold (Km = 0.8 +/- 0.08 microM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 micro M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with Km values of 79 and 960 microM, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor (Km = 1100 +/- 140 microM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on Mg2+, and activity was stimulated by potassium, as reflected by a decrease in the Km and an increase in Vmax for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.     

Molecular characterization of Mycobacterium tuberculosis isolates from Izmir, Turkey

In recent years, molecular typing methods have been used in epidemiologic studies of Mycobacterium tuberculosis isolates in various areas of the world. However, there have been few data on this issue in Turkey. We describe the molecular characterization of 56 Mycobacterium tuberculosis isolates recovered from individual patients in Izmir and the surrounding area by three different molecular methods. Isolated M. tuberculosis strains were characterized by IS6110 RFLP, spoligotyping and major genetic group designation. In total, 51 RFLP and 35 spoligopatterns were identified. Fourteen (25%) isolates were indicated as low copy number. Based on three genotypic characterization methods together, five clusters with two isolates each were identified. Most of the isolates (98.2%) were assigned as genetic groups 2 or 3. Only one isolate was identified as Beijing family strain (principal genetic group 1). The shared international clades were found to be Beijing-family, var T1 (ST 37), LAM (Latin-American-Mediterranean) 7 (ST 41), LAM 9 (ST 42), Haarlem 1 (ST 47), Haarlem 3 (ST 50) and T1 (ST 53). In this study, IS6110 RFLP, spoligotyping and major genetic group designation were found to be useful methods for molecular epidemiologic studies.     

Purine nucleoside phosphorylase from Mycobacterium tuberculosis. Analysis of inhibition by a transition-state analogue and dissection by parts.

Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP). The gene was cloned, expressed, purified, and found to exhibit PNP activity. Purified M. tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Escherichia coli enzyme. Immucillin-H is a rationally designed analogue of the transition state that has been shown to be a potent inhibitor of mammalian PNP's. This inhibitor also exhibits slow-onset inhibition of M. tuberculosis PNP with a rapid, reversible inhibitor binding (K(i) of 2.2 nM) followed by an overall dissociation constant (K(i)) of 28 pM, yielding a K(m)/K(i) value of 10(6). Time-dependent tight binding of the inhibitor occurs with a rate of 0.1 s(-)(1), while relaxation of the complex is slower at 1.4 x 10(-)(3) s(-)(1). The pH dependence of the K(i) value of immucillin-H to the M. tuberculosis PNP suggests that the inhibitor binds as the neutral, unprotonated form that is subsequently protonated to generate the tight-binding species. The M. tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP. Analysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K(is) of 0.39 microM) while the 1,4-dideoxy-1,4-iminoribitol binds weakly (K(is) of 2.9 mM). Double-inhibition studies demonstrate antagonistic binding of 9-deazahypoxanthine and iminoribitol (beta = 13). However, the covalent attachment of these two components in immucillin-H increases equilibrium binding affinity by a factor of >14 000 (28 pM vs 0.39 microM) compared to 9-deazahypoxanthine alone, and by a factor of >10(8) compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements. The structural basis for M. tuberculosis PNP inhibition by immucillin-H and by its component parts is reported in the following paper [Shi, W., Basso, L. A., Santos, D. S., Tyler, P. C., Furneaux, R. H., Blanchard, J. S., Almo, S. C., and Schramm, V. L. (2001) Biochemistry 40, 8204-8215].     

Biophysical characterization and ligand-binding properties of the elongation factor Tu from Mycobacterium tuberculosis

Mycobacterium tuberculosis(Mtb)is the key devastating bacterial pathogen responsible for tuberculosis.Increasing emergence of multi-drug-resistant,extensively drug-resistant,and rifampicin/isoniazid-resistant strains of Mtb makes the discovery of validated drug targets an urgent priority.As a vital translational component of the protein biosynthesis system,elongation factor Tu(EF-Tu)is an important molecular switch responsible for selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome.In addition,EF-Tu from Mtb(MtbEF-Tu)is involved in the initial step of trans-translation which is an effective system for rescuing the stalled ribosomes from non-stop translation complexes under stress conditions.Given its crucial role in protein biosynthesis,EF-Tu is identified as an excellent molecular target for drug design.Here,we reported the recombinant expression,purification,biophysical characterization,and structural modeling of the MtbEF-Tu protein.Our results demonstrated that prokaryotic expression plasmids of pET28a-MtbEF-Tu could be expressed efficiently in Escherichia coli.We successfully purified the 6× His-tagged proteins with a yield of 16.8 mg from 1 l of Luria Bertani medium.Dynamic light scattering experiments showed that MtbEF-Tu existed in a monomeric form,and circular dichroism experiments indicated that MtbEF-Tu was well structured.Moreover,isothermal titration calorimetry experiments displayed that the purified MtbEF-Tu protein possessed intermediate binding affinities for guanosine-5′-triphosphate(GTP)and GDP.The GTP/GDP-binding sites were predicted by flexible molecular docking approach which reveals that GTP/GDP binds to MtbEF-Tu mainly through hydrogen bonds.Our work lays the essential basis for further structural and functional studies of MtbEF-Tu as well as MtbEF-Tu-related novel drug developments.     

Expression and characterization of the carboxyl esterase Rv3487c from Mycobacterium tuberculosis

Rv3487c (lipF), a member of the lipase family of Mycobacterium tuberculosis, is related to virulence of this pathogen. Real-time RT-PCR analysis indicated that Rv3487c was induced at low pH in M. tuberculosis cultured in vitro. The gene of Rv3487c was cloned and expressed as fusion protein in Escherichia coli. After removal of the N-terminal domain of the fusion partner by enterokinase treatment, the effect of pH, temperature, and detergents on the purified enzyme activity and stability was characterized. Rv3487c could efficiently hydrolyze short chain esters. The catalytic triad of Rv3487c consists of residues Ser90, Glu189, and His219 as demonstrated by amino acid sequence alignment, three-dimensional modeling, and site-directed mutagenesis.     

Cloning and characterization of aspartate-beta-semialdehyde dehydrogenase from Mycobacterium tuberculosis H37 Rv

AIMS: To clone and characterize the aspartate-beta-semialdehyde dehydrogenase of Mycobacterium tuberculosis H37Rv. METHODS AND RESULTS: The asd gene of M. tuberculosis H37Rv was cloned in pGEM-T Easy vector, subcloned in expression vector pQE30 having a T5 promoter, and overexpressed in Escherichia coli. The ASD enzyme was expressed to levels of 40% but was found to be inactive. Functional ASD was obtained by altering induction and growth conditions and the enzyme was purified to near homogeneity using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The K(m) and V(max) values for the three substrates L-ASA, NADP and Pi, the turnover number and specific activity of the enzyme were determined. CONCLUSIONS: Functional ASD enzyme of M. tuberculosis was obtained by gene cloning and protein purification using affinity chromatography. The K(cat) and specific activity of the enzyme were 8.49 s(-1) and 13.4 micromol min(-1) microg(-1) respectively. Significance and Impact of the Study: The ASD enzyme is a validated drug target. We characterized this enzyme from M. tuberculosis and future work would focus on deducing the three-dimensional structure of the enzyme and design of inhibitors, which could be used as drugs against TB.     

Expression,purification and characterization of UvrD2 helicase from Mycobacterium tuberculosis

Helicases appear to be important for genome stability of dormant Mycobacterium tuberculosis responsible for latent tuberculosis infection and big proportion of active disease cases caused by reactivation. It was demonstrated that in both M. tuberculosis and Mycobacterium smegmatis, a helicase termed UvrD2 is essential for bacterial growth making it a promising target to fight tuberculosis. In many cases expression of soluble and active mycobacterial proteins in Escherichia coli is a complicated issue. In this work we for the first time report a non-trivial expression procedure in E. coli, leading to soluble UvrD2 from M. tuberculosis which possesses DNA-dependent ATP-ase activity.     

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diaminopelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75 +/- 0.11 M-1s-1. The Km values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 microM and 308.28 microM, respectively whereas the Vmax and kcat values for the enzyme are 0.02074 micromoles/min/ml and 0.003 s-1, respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme.     

Electrochemical characterization of mutant forms of rubredoxin B from Mycobacterium tuberculosis

Electron transfer in metalloproteins is a driving force for many biological processes and widely distributed in nature. Rubredoxin B (RubB) from Mycobacterium tuberculosis is a first example among [1Fe-0S] proteins that support catalytic activity of terminal sterol-monooxygenases enabling its application in metabolic engineering. To explore the tolerance of RubB to the specific amino acid changes we evaluated the effect of surface mutations on its electrochemical properties. Based on the RubB fold we also designed the mutant with a putative additional site for protein-protein interactions to further evaluate electron transfer and electrochemical properties. The investigation of redox properties of mutant variants of RubB was done using screen-printed graphite electrodes (SPEs) modified with stable dispersion of multi-walled carbon nanotubes (MWCNTs). The redox potentials (midpoint potentials, E^0?) of mutants did not significantly differ from the wild type protein and vary in the range of ?264 to ?231 mV vs. Ag/AgCl electrode. However, all mutations affect electron transfer rate between the protein and electrode. Notably, the modulation of the protein-protein interactions was observed for the insertion mutant suggesting the possibility of tailoring of rubredoxin for the selected redox-partner. Overall, RubB is tolerant to the significant modifications in its structure enabling rational engineering of novel redox proteins.