Delving into the Characteristic Features of "Menace" Mycobacterium tuberculosis Homologs: A Structural Dynamics and Proteomics Perspectives.

The global increase in the morbidity/mortality rate of Mycobacterial infections, predominantly renascent tuberculosis, leprosy, and Buruli ulcers have become worrisome over the years. More challenging is the incidence of resistance mediated by mutant Mycobacterium strains against front-line antitubercular drugs. Homologous to all Mycobacteria species is the GlcNAc-6-phosphate deacetylase (NagA) which catalyzes essential amino sugars synthesis required for cell wall architecture, hence, metamorphosing into an important pharmacological target for curtailing virulence and drug-resistance. This study used integrated bioinformatics methods, MD simulations, and DynaMut and PolyPhen2 to; explore unique features, monitor dynamics, and analyze the functional impact of non-synonymous single-nucleotide polymorphisms of the six NagA of most ruinous Mycobacterium species; tuberculosis (Mtb), smegmatis (MS), marinum (MM), ulcerans, africanum, and microti respectively. This approach is essential for multi-targeting and could result in the identification of potential polypharmacological antitubercular compounds. Comparative sequential analyses revealed ≤ 50% of the overall structure, including the catalytic Asp267 and reactive Cys131, remained conserved. Interestingly, MS-NagA and MM-NagA possess unique hydrophobic isoleucine (Ile) residues at their active sites in contrast to leucine (Leu) found in other variants. More so, unique to the active sites of the NagA is a 'subunit loop' that covers the active site; probably crucial in binding (entry and exit) mechanisms of targeted NagA inhibitors. Relatively, nsSNP mutations exerted a destabilizing effect on the native NagA conformation. Structural and dynamical insights provided, basically pin-pointed the "Achilles' heel" explorable for the rational drug design of target-specific 'NagA' inhibitors potent against a wide range of mycobacterial diseases.

Arginine-95 is important for recruiting superoxide to the active site of the FerB flavoenzyme of Paracoccus denitrificans.

Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.

Drug targeted virtual screening and molecular dynamics of LipU protein of Mycobacterium tuberculosis and Mycobacterium leprae.

The lipolytic protein LipU was conserved in mycobacterium sp. including M. tuberculosis (MTB LipU) and M. leprae (MLP LipU). The MTB LipU was identified in extracellular fraction and was reported to be essential for the survival of mycobacterium. Therefore to address the problem of drug resistance in pathogen, LipU was selected as a drug target and the viability of finding out some FDA approved drugs as LipU inhibitors in both the cases was explored. Three-dimensional (3D) model structures of MTB LipU and MLP LipU were generated and stabilized through molecular dynamics (MD). FDA approved drugs were screened against these proteins. The result showed that the top-scoring compounds for MTB LipU were Diosmin, Acarbose and Ouabain with the Glide XP score of -12.8, -11.9 and -11.7 kcal/mol, respectively, whereas for MLP LipU protein, Digoxin (-9.2 kcal/mol), Indinavir (-8.2 kcal/mol) and Travoprost (-8.2 kcal/mol) showed highest affinity. These drugs remained bound in the active site pocket of MTB LipU and MLP LipU structure and interaction grew stronger after dynamics. RMSD, RMSF and Rg were found to be persistent throughout the simulation period. Hydrogen bonds along with large number of hydrophobic interactions stabilized the complex structures. Binding free energies obtained through Prime/MM-GBSA were found in the significant range from -63.85 kcal/mol to -34.57 kcal/mol for MTB LipU and -71.33 kcal/mol to -23.91 kcal/mol for MLP LipU. The report suggested high probability of these drugs to demolish the LipU activity and could be probable drug candidates to combat TB and leprosy disease.

Characterization of Ofloxacin Interaction with Mutated (A91V) Quinolone Resistance Determining Region of DNA Gyrase in Mycobacterium Leprae through Computational Simulation.

Mycobacterium leprae, the causal agent of leprosy is non-cultivable in vitro. Thus, the assessment of antibiotic activity against Mycobacterium leprae depends primarily upon the time-consuming mouse footpad system. The GyrA protein of Mycobacterium leprae is the target of the antimycobacterial drug, Ofloxacin. In recent times, the GyrA mutation (A91V) has been found to be resistant to Ofloxacin. This phenomenon has necessitated the development of new, long-acting antimycobacterial compounds. The underlying mechanism of drug resistance is not completely known. Currently, experimentally crystallized GyrA-DNA-OFLX models are not available for highlighting the binding and mechanism of Ofloxacin resistance. Hence, we employed computational approaches to characterize the Ofloxacin interaction with both the native and mutant forms of GyrA complexed with DNA. Binding energy measurements obtained from molecular docking studies highlights hydrogen bond-mediated efficient binding of Ofloxacin to Asp47 in the native GyrA-DNA complex in comparison with that of the mutant GyrA-DNA complex. Further, molecular dynamics studies highlighted the stable binding of Ofloxacin with native GyrA-DNA complex than with the mutant GyrA-DNA complex. This mechanism provided a plausible reason for the reported, reduced effect of Ofloxacin to control leprosy in individuals with the A91V mutation. Our report is the first of its kind wherein the basis for the Ofloxacin drug resistance mechanism has been explored with the help of ternary Mycobacterium leprae complex, GyrA-DNA-OFLX. These structural insights will provide useful information for designing new drugs to target the Ofloxacin-resistant DNA gyrase.

Characterization of ML0314c of Mycobacterium leprae and deciphering its role in the immune response in leprosy patients.

Mycobacterium leprae has a reduced genome size due to the reductive evolution over a long period of time. Lipid metabolism plays an important role in the life cycle and pathogenesis of this bacterium. In comparison to 26 lip genes (Lip A-Z) of M. tuberculosis, M. leprae retained only three orthologs indicating their importance in its life cycle. ML0314c (LipU) is one of them. It is conserved throughout the mycobacterium species. Bioinformatics analysis showed the presence of an α/ß hydrolase fold and 'GXSXG' characteristic of the esterases/lipases. The gene was expressed in E. coli and purified to homogeneity. It showed preference towards short chain esters with pNP-acetate as the preferred substrate. The enzyme showed optimal activity at 45°C and pH8.0. ML0314c protein was stable between temperatures ranging from 20 to 60°C and pH5.0-8.0, i.e., relatively acidic and neutral conditions. The active site residues predicted bioinformatically were confirmed to be Ser168, Glu267, and His297 by site directed mutagenesis. E-serine, DEPC and Tetrahydrolipstatin (THL) completely inhibited the activity of ML0314c. The protein was localized in cell wall and extracellular medium. Several antigenic epitopes were predicted in ML0314c. Protein elicited strong humoral immune response in leprosy patients, whereas, a reduced immune response was observed in the relapsed cases. No humoral response was observed in treatment completed patients. Overexpression of ml0314c in the surrogate host M. smegmatis showed marked difference in the colony morphology and growth rate. In conclusion, ML0314c is a secretary carboxyl esterase that could modulate the immune response in leprosy patients.

Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution.

Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into ß-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.

AcsA-AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769.

The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.

Structural studies suggest a peptidoglycan hydrolase function for the Mycobacterium tuberculosis Tat-secreted protein Rv2525c.

Among the few proteins shown to be secreted by the Tat system in Mycobacterium tuberculosis, Rv2525c is of particular interest, since its gene is conserved in the minimal genome of Mycobacterium leprae. Previous evidence linked this protein to cell wall metabolism and sensitivity to ß-lactams. We describe here the crystal structure of Rv2525c that shows a TIM barrel-like fold characteristic of glycoside hydrolases of the GH25 family, which includes prokaryotic and phage-encoded peptidoglycan hydrolases. Structural comparison with other members of this family combined with substrate docking suggest that, although the 'neighbouring group' catalytic mechanism proposed for this family still appears as the most plausible, the identity of residues involved in catalysis in GH25 hydrolases might need to be revised.

Exploring the molecular basis for selective binding of homoserine dehydrogenase from Mycobacterium leprae TN toward inhibitors: a virtual screening study.

Homoserine dehydrogenase (HSD) from Mycobacterium leprae TN is an antifungal target for antifungal properties including efficacy against the human pathogen. The 3D structure of HSD has been firmly established by homology modeling methods. Using the template, homoserine dehydrogenase from Thiobacillus denitrificans (PDB Id 3MTJ), a sequence identity of 40% was found and molecular dynamics simulation was used to optimize a reliable structure. The substrate and co-factor-binding regions in HSD were identified. In order to determine the important residues of the substrate (L-aspartate semialdehyde (L-ASA)) binding, the ASA was docked to the protein; Thr163, Asp198, and Glu192 may be important because they form a hydrogen bond with HSD through AutoDock 4.2 software. neuraminidaseAfter use of a virtual screening technique of HSD, the four top-scoring docking hits all seemed to cation-π ion pair with the key recognition residue Lys107, and Lys207. These ligands therefore seemed to be new chemotypes for HSD. Our results may be helpful for further experimental investigations.

Co-factor binding confers substrate specificity to xylose reductase from Debaryomyces hansenii.

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards "non-substrate" sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K(d)∼5.0-10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4-5 fold molar excess. Comparison of K(d) values with K(m) values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.

Loop residues and catalysis in OMP synthase.

Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.

Homology modeling and docking analyses of M. leprae Mur ligases reveals the common binding residues for structure based drug designing to eradicate leprosy.

Multi drug resistance capacity for Mycobacterium leprae (MDR-Mle) demands the profound need for developing new anti-leprosy drugs. Since most of the drugs target a single enzyme, mutation in the active site renders the antibiotic ineffective. However, structural and mechanistic information on essential bacterial enzymes in a pathway could lead to the development of antibiotics that targets multiple enzymes. Peptidoglycan is an important component of the cell wall of M. leprae. The biosynthesis of bacterial peptidoglycan represents important targets for the development of new antibacterial drugs. Biosynthesis of peptidoglycan is a multi-step process that involves four key Mur ligase enzymes: MurC (EC:6.3.2.8), MurD (EC:6.3.2.9), MurE (EC:6.3.2.13) and MurF (EC:6.3.2.10). Hence in our work, we modeled the three-dimensional structure of the above Mur ligases using homology modeling method and analyzed its common binding features. The residues playing an important role in the catalytic activity of each of the Mur enzymes were predicted by docking these Mur ligases with their substrates and ATP. The conserved sequence motifs significant for ATP binding were predicted as the probable residues for structure based drug designing. Overall, the study was successful in listing significant and common binding residues of Mur enzymes in peptidoglycan pathway for multi targeted therapy.

An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae.

CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 µM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 µM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.

From Zn to Mn: the study of novel manganese-binding groups in the search for new drugs against tuberculosis.

In most eubacteria, apicomplexans, and most plants, including the causal agents for diseases such as malaria, leprosy, and tuberculosis, the methylerythritol phosphate pathway is the route for the biosynthesis of the C(5) precursors to the essential isoprenoid class of compounds. Owing to their absence in humans, the enzymes of the methylerythritol phosphate pathway have become attractive targets for drug discovery. This work investigates a new class of inhibitors against the second enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Inhibition of this enzyme may involve the chelation of a crucial active site Mn ion, and the metal-chelating moieties studied here have previously been shown to be successful in application to the zinc-dependent metalloproteinases. Quantum mechanics and docking calculations presented in this work suggest the transferability of these metal-chelating compounds to Mn-containing 1-deoxy-D-xylulose 5-phosphate reductoisomerase enzyme, as a promising starting point to the development of potent inhibitors.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not.

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Granulosain I, a cysteine protease isolated from ripe fruits of Solanum granuloso-leprosum (Solanaceae).

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.

Characterization of phylogenetically distant members of the adenylate cyclase family from mycobacteria: Rv1647 from Mycobacterium tuberculosis and its orthologue ML1399 from M. leprae.

Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylate and guanylate cyclases. Rv1647 was predicted to be an active adenylate cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, the cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the previously characterized Rv1625c adenylate cyclase. Adenylate cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate-specifying residues to those present in guanylate cyclases failed to convert the enzyme into a guanylate cyclase, and did not alter its oligomeric status. Orthologues of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The orthologue from M. leprae (ML1399) was cloned, and the protein was expressed, purified and shown biochemically to be an adenylate cyclase, thus representing the first adenylate cyclase to be described from M. leprae. Importantly, Western-blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that the Rv1647 and ML1399 gene products respectively were expressed in these bacteria. Additionally, M. tuberculosis was also found to express the Rv1625c adenylate cyclase, suggesting that multiple adenylate cyclase proteins may be expressed simultaneously in this organism. These results suggest that class III cyclase-like gene products probably have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.

Crystal structure of the catalytic domain of the PknB serine/threonine kinase from Mycobacterium tuberculosis.

With the advent of the sequencing programs of prokaryotic genomes, many examples of the presence of serine/threonine protein kinases in these organisms have been identified. Moreover, these kinases could be classified as homologues of those belonging to the well characterized superfamily of the eukaryotic serine/threonine and tyrosine kinases. Eleven such kinases were recognized in the genome of Mycobacterium tuberculosis. Here we report the crystal structure of an active form of PknB, one of the four M. tuberculosis kinases that are conserved in the downsized genome of Mycobacterium leprae and are therefore presumed to play an important role in the processes that regulate the complex life cycle of mycobacteria. Our structure confirms again the extraordinary conservation of the protein kinase fold and constitutes a landmark that extends this conservation across the evolutionary distance between high eukaryotes and eubacteria. The structure of PknB, in complex with a nucleotide triphosphate analog, reveals an enzyme in the active state with an unprecedented arrangement of the Gly-rich loop associated with a new conformation of the nucleotide gamma-phosphoryl group. It presents as well a partially disordered activation loop, suggesting an induced fit mode of binding for the so far unknown substrates of this kinase or for some modulating factor(s).

Modulation of the catalytic activity of neutrophil collagenase MMP-8 on bovine collagen I. Role of the activation cleavage and of the hemopexin-like domain.

The cleavage of bovine collagen I by neutrophil collagenase MMP-8 has been followed at pH 7.4, 37 degrees C. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990) Biochemistry 29, 10628-10634; Knäuper, V., Osthues, A., DeClerk, Y. A., Langley, K. A., Bläser, J., and Tschesche, H. (1993) Biochem. J. 291, 847-854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657-18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met(80) of the proenzyme (MetMMP-8) and the former Phe(79) of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much faster k(cat)/K(m), fully attributable to a lower K(m), suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227-233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.

The crystal structure and active site location of isocitrate lyase from the fungus Aspergillus nidulans.

BACKGROUND: Isocitrate lyase catalyses the first committed step of the carbon-conserving glyoxylate bypass, the Mg(2+)-dependent reversible cleavage of isocitrate into succinate and glyoxylate. This metabolic pathway is an inviting target for the control of a number of diseases, because the enzymes involved in this cycle have been identified in many pathogens including Mycobacterium leprae and Leishmania. RESULTS: As part of a programme of rational drug design the structure of the tetrameric Aspergillus nidulans isocitrate lyase and its complex with glyoxylate and a divalent cation have been solved to 2.8 A resolution using X-ray diffraction. Each subunit comprises two domains, one of which adopts a folding pattern highly reminiscent of the triose phosphate isomerase (TIM) barrel. A 'knot' between subunits observed in the three-dimensional structure, involving residues towards the C terminus, implies that tetramer assembly involves considerable flexibility in this part of the protein. CONCLUSIONS: Difference Fourier analysis together with the pattern of sequence conservation has led to the identification of both the glyoxylate and metal binding sites and implicates the C-terminal end of the TIM barrel as the active site, which is consistent with studies of other enzymes with this fold. Two disordered regions of the polypeptide chain lie close to the active site, one of which includes a critical cysteine residue suggesting that conformational rearrangements are essential for catalysis. Structural similarities between isocitrate lyase and both PEP mutase and enzymes belonging to the enolase superfamily suggest possible relationships in aspects of the mechanism.