Structural analysis reveals DNA binding properties of Rv2827c, a hypothetical protein from Mycobacterium tuberculosis

Tuberculosis (TB) is a major global health threat caused by Mycobacterium tuberculosis (Mtb). It is further fueled by the HIV pandemic and by increasing incidences of multidrug resistant Mtb-strains. Rv2827c, a hypothetical protein from Mtb, has been implicated in the survival of Mtb in the macrophages of the host. The three-dimensional structure of Rv2827c has been determined by the three-wavelength anomalous diffraction technique using bromide-derivatized crystals and refined to a resolution of 1.93 Å. The asymmetric unit of the orthorhombic crystals contains two independent protein molecules related by a non-crystallographic translation. The tertiary structure of Rv2827c comprises two domains: an N-terminal domain displaying a winged helix topology and a C-terminal domain, which appears to constitute a new and unique fold. Based on structural homology considerations and additional biochemical evidence, it could be established that Rv2827c is a DNA-binding protein. Once the understanding of the structure-function relationship of Rv2827c extends to the function of Rv2827c in vivo, new clues for the rational design of novel intervention strategies may be obtained.     

DNA polymerase III α subunit from Mycobacterium tuberculosis H37Rv: Homology modeling and molecular docking of its inhibitor

The alpha subunit of Mycobacterial DNA polymerase III holo enzyme catalyzes the polymerization of both DNA strands. The present investigation reports three dimensional (3-D) structure model of DNA polymerase III α subunit of Mycobacterium tuberculosis H37Rv (MtbDnaE1) generated using homology modeling with the backbone structure of DNA polymerase III α of Thermus aquaticus as a template. The model was evaluated at various structure verification servers, which assess the stereo chemical parameters of the residues in the model, as well as structural and functional domains. Comparative analysis of MtbDnaE1 structure reveals the structure of its catalytic domain to be unrelated to that of the human. Successful docking of known inhibitor of bacterial DNA polymerases, 251D onto the modeled MtbDnaE1 was also performed. Therefore, the structure model of MtbDnaE1, a potential anti-mycobacterial target, opens a new avenue for structure-based drug designing against the pathogen. ABBREVIATIONS: aa - amino acid(s), PolIIIα - DNA polymerase III alpha subunit, Taq Pol IIIα - Pol IIIα of Thermus aquaticus, MtbDnaE1 - PolIIIα of Mycobacterium tuberculosis.     

Genomic imprinting in mammals: an interplay between chromatin and DNA methylation?

Most imprinted loci have key regulatory elements that are methylated on only one of the parental chromosomes. For several of these 'differentially methylated regions', recent studies establish that the unmethylated chromosome has a specialized chromatin organization that is characterized by nuclease hypersensitivity. The novel data raise the question of whether specific proteins and associated chromatin features regulate the allele-specificity of DNA methylation at these imprinting control elements.     

Carbonic anhydrase inhibitors. Characterization and inhibition studies of the most active β-carbonic anhydrase from Mycobacterium tuberculosis,Rv3588c

The Rv3588c gene product of Mycobacterium tuberculosis, a β-carbonic anhydrase (CA, EC 4.2.1.1) denominated here mtCA 2, shows the highest catalytic activity for CO₂ hydration (k_cat of 9.8 × 10⁵ s^?1, and k_cat/K_m of 9.3 × 10⁷ M^?1 s^?1) among the three β-CAs encoded in the genome of this pathogen. A series of sulfonamides/sulfamates was assayed for their interaction with mtCA 2, and some diazenylbenzenesulfonamides were synthesized from sulfanilamide/metanilamide by diazotization followed by coupling with amines or phenols. Several low nanomolar mtCA 2 inhibitors have been detected among which acetazolamide, ethoxzolamide and some 4-diazenylbenzenesulfonamides (K_Is of 9–59 nM). As the Rv3588c gene was shown to be essential to the growth of M. tuberculosis, inhibition of this enzyme may be relevant for the design of antituberculosis drugs possessing a novel mechanism of action.     

Evaluation of sulphonamide derivatives acting as inhibitors of human carbonic anhydrase isoforms I,II and Mycobacterium tuberculosis β-class enzyme Rv3273

A series of novel sulphonamide derivatives was obtained from sulphanilamide which was N4-alkylated with ethyl bromoacetate followed by reaction with hydrazine hydrate. The hydrazide obtained was further reacted with various aromatic aldehydes. The novel sulphonamides were characterised by infrared, mass spectrometry, ¹H- and ¹³C-NMR and purity was determined by high-performance liquid chromatography (HPLC). Human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I and II and Mycobacterium tuberculosis β-CA encoded by the gene Rv3273 (mtCA 3) inhibition activity was investigated with the synthesised compounds which showed promising inhibition. The K_Is were in the range of 54.6?nM–1.8?µM against hCA I, in the range of 32.1?nM–5.5?µM against hCA II and of 127?nM–2.12?µM against mtCA 3.     

Characterization of the Corynebacterium glutamicum ΔpimB′ ΔmgtA Double Deletion Mutant and the Role of Mycobacterium tuberculosis Orthologues Rv2188c and Rv0557 in Glycolipid Biosynthesis

In this study, utilizing a Corynebacterium glutamicum ΔpimB′ ΔmgtA double deletion mutant, we unequivocally assign the in vivo functions of Rv2188c as an Ac₁PIM₁:mannosyltransferase (originally termed PimB′_Mt [Mycobacterium tuberculosis PimB′]) and Rv0557 as a GlcAGroAc₂:mannosyltransferase (originally termed PimB_Mt), which we have reassigned as PimB_Mt and MgtA_Mt, respectively, in Mycobacterium tuberculosis.The current model of mycobacterial phosphatidyl-myo-inositol mannoside (PIM) biosynthesis, supported by biochemical and genetic studies, follows a linear pathway from phosphatidylinositol (PI) → Ac₁PIM₂ → Ac₁PIM₄ → Ac₁PIM₆ (4, 17, 19) as shown in Fig. ​Fig.1.1. In this pathway, mycobacterial PI is glycosylated by an α-mannopyranosyl residue at the 2-OH position of inositol, followed by the acylation and mannosylation at the 6-OH position of PI to form Ac₁PIM₂ (3), which is further mannosylated to form Ac₁PIM₄ and Ac₁PIM₆, extending the 6-OH position of Ac₁PIM₂ (19).Open in a separate windowFIG. 1.Glycolipid biosynthetic pathways in Corynebacterineae. (A) PIM synthesis in M. tuberculosis; (B) PIMs; (C) ManGlcAGroAc₂ synthesis in C. glutamicum.In view of the identification of genes involved in PIM, lipomannan (LM), and lipoarabinomannan (LAM) biosynthesis, Schaeffer et al. (22) proposed Rv0557 as an α-d-mannose-α-(1→6)-phosphatidyl-myo-inositol-mannosyltransferase that transfers mannose from GDP-Man to Ac₁PIM₁ to form Ac₁PIM₂, a precursor of the immunomodulatory lipoglycans LM and LAM (4, 17). The study was based on a cell-free assay using GDP[¹⁴C]Man, Ac₁PIM_1, Mycobacterium smegmatis membranes, and/or partially purified recombinant Rv0557. On the basis of these in vitro studies, Rv0557 was assigned as PimB_Mt (Mycobacterium tuberculosis PimB) in the synthesis of Ac₁PIM₂. However, on the disruption of Rv0557 in Mycobacterium tuberculosis, PIM biosynthesis remains unaffected (G. S. Besra and L. S. Schlesinger, unpublished data), suggesting that either gene duplication or Rv0557 performed another function in M. tuberculosis. Interestingly, in a recent study, Rv0557 was also shown to be involved in the biosynthesis of 1,2-di-O-C₁₆/C_18:1-(α-d-mannopyranosyl)-(1→4)-(α-d-glucopyranosylu- ronic acid)-(1→3)-glycerol (ManGlcAGroAc₂) and an LM-like molecule in Corynebacterium glutamicum and was termed MgtA_Mt (M. tuberculosis MgtA) (25). More recently, Rv2188c was also proposed to be involved in the synthesis of Ac₁PIM₂ as the second α-d-mannose-α-(1→6)-phosphatidyl-myo-inositol-mannosyl transferase (termed PimB′_Mt) (13, 16), which has augmented ongoing confusion in the field. Due to the essentiality of M. tuberculosis PIM biosynthesis (3) in this study, we have generated C. glutamicum ΔpimB′ ΔmgtA, deficient in pimB′_Cg and mgtA_Cg (C. glutamicum pimB′ and mgtA) and subsequently overexpressed Rv2188c and Rv0557 individually to identify their true in vivo and in vitro biochemical activities.     

Replacement of cardiotoxic aminopiperidine linker with piperazine moiety reduces cardiotoxicity? Mycobacterium tuberculosis novel bacterial topoisomerase inhibitors

Recently numerous non-fluoroquinolone-based bacterial type II topoisomerase inhibitors from both the GyrA and GyrB classes have been reported as antibacterial agents. Inhibitors of the GyrA class include aminopiperidine-based novel bacterial type II topoisomerase inhibitors (NBTIs). However, inhibition of the cardiac ion channel remains a serious liability for the aminopiperidine based NBTIs. In this paper we replaced central aminopiperidine linker with piperazine moiety and tested for its biological activity. We developed a series of twenty four compounds with a piperazine linker 1-(2-(piperazin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one, by following a multistep protocol. Among them compound 4-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)-N-(4-nitrophenyl)piperazine-1-carboxamide (11) was the most promising inhibitor with Mycobacterium tuberculosis (MTB) DNA gyrase enzyme supercoiling IC₅₀ of 0.29 ± 0.22 μM, with a good MTB MIC of 3.45 μM. These kind of compounds retains good potency and showed reduced cardiotoxicity compared to aminopiperidines.     

Ribonucleotide reductases: the link between an RNA and a DNA world?

The structures of a class III ribonucleotide reductase (RNR) and pyruvate formate lyase exhibit striking homology within their active site domains with respect to each other and to the previously published structure of a class I RNR. The common structures and the common complex-radical-based chemistry of these systems, as well as of the class II RNRs, suggest that RNRs evolved by divergent evolution and provide an essential link between the RNA and DNA world.     

Does topoisomerase II specifically recognize and cleave hairpins,cruciforms and crossovers of DNA?

DNA topoisomerase II is an enzyme that specializes in DNA disentanglement. It catalyzes the interconversion of DNA between different topological states. This event requires the passage of one duplex through another one via a transient double-strand break. Topoisomerase II is able to process any type of DNA, including structures such as DNA juxtapositions (crossovers), DNA hairpins or cruciforms, which are recognized with high specificity. In this review, we focused our attention on topoisomerase II recognizing DNA substrates that possess particular geometries. A strong cleavage site, as we identified in pBR322 DNA in the presence of ellipticine (site 22), appears to be characterized by a cruciform structure formed from two stable hairpins. The same sequence could also constitute a four-way junction structure stabilized by interactions involving ATC sequences. The latter have been shown to be able to promote Holliday junctions. We reviewed the recent literature that deals with the preferential recognition of crossovers by various topoisomerases. The single molecule relaxation experiments have demonstrated the differential abilities of the topoisomerases to recognize crossovers. It appears that enzymes, which distinguish the chirality of the crossovers, possess specialized domains dedicated to this function. We also stress that the formation of crossovers is dependent on the presence of adequate stabilizing sequences. Investigation of the impact of such structures on enzyme activity is important in order to both improve our knowledge of the mechanism of action of the topoisomerase II and to develop new inhibitors of this enzyme.     

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.     

α-Carotene and its derivatives have a sole chirality in phototrophic organisms?

Carotenoids in eukaryotic phototrophic organisms can be classified into two groups; β-carotene and its derivatives, and α-carotene and its derivatives. We re-examined distribution of α-carotene and its derivatives among various taxa of aquatic algae (17 classes) and land plants. α-carotene and its derivatives were found from Rhodophyceae (macrophytic type), Cryptophyceae, Euglenophyceae, Chlorarachniophyceae, Prasinophyceae, Chlorophyceae, Ulvophyceae, Charophyceae, and land plants, while they could not be detected from Glaucophyceae, Rhodophyceae (unicellular type), Chryosophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, Xanthophyceae, Eustigmatophyceae, Haptophyceae, and Dinophyceae. We also analyzed the chirality of α-carotene and/or its derivatives, such as lutein and siphonaxanthin, and found all of them had only (6'R)-type, not (6'S)-type.     

Purification and molecular characterization of a novel diadenosine 5′,5′′′-P1,P4-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv

In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5′-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5′-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the K_m values for Ap4A and phosphate were 0.10 and 0.94 mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif.     

Co-localization of chicken DNA topoisomerase IIα, but not β, with sites of DNA replication and possible involvement of a C-terminal region of α through its binding to PCNA

Clones for DNA topoisomerase IIalpha and beta (topo-IIalpha and beta) were isolated from a cDNA expression library of chicken MSB-1 cells by immunoscreening. The deduced sequences of chicken topo-IIalpha and beta were about 80% identical for the N-terminal ATPase domain and the central core domain but only 37% for the C-terminal domain. Polyclonal antibodies were raised against C-terminal polypeptides specific to topo-IIalpha and beta. Indirect immunofluorescence with these antibodies to chicken embryonic fibroblasts demonstrated that topo-IIalpha was distributed in discrete intranuclear spots, which coincided with sites of DNA replication as indicated by incorporation of 5-bromo-2'-deoxyuridine, whereas topo-IIbeta was distributed rather uniformly within a nucleus. Examination of intranuclear distribution patterns of chimeric constructs between topo-IIalpha and beta suggested that a sequence region (residues 1280-1294) in the C-terminal domain of topo-IIalpha was effective in co-localization with sites of DNA replication. This region consists of a QTxhxF motif (x, any residue; h, hydrophobic residue) followed by a KR-rich sequence, which resembles those found in several proteins known to associate with proliferating cell nuclear antigen (PCNA) or targeted to the replication factory. An in vitro pull-down assay with glutathione-S-transferase-PCNA and (His)6-tagged truncated forms of topo-IIalpha demonstrated that polypeptides containing the above region (residues 1158-1553 or 1158-1294) bound to PCNA in vitro.     

Identification of 2-Aminothiazole-4-Carboxylate Derivatives Active against Mycobacterium tuberculosis H37Rv and the β-Ketoacyl-ACP Synthase mtFabH

Background

Tuberculosis (TB) is a disease which kills two million people every year and infects approximately over one-third of the world''s population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration.

Methodology/Principal Findings

Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM) to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM''s novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H₃₇R_v and, dissociatively, against the β-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H₃₇R_v with an MIC of 0.06 µg/ml (240 nM), but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)thiazole-4-carboxylate inhibited mtFabH with an IC₅₀ of 0.95±0.05 µg/ml (2.43±0.13 µM) but was not active against the whole cell organism.

Conclusions/Significance

These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents.     

Synthesis and biological evaluation of substituted 4-arylthiazol-2-amino derivatives as potent growth inhibitors of replicating Mycobacterium tuberculosis H₃₇Rv

In search of potential therapeutics for tuberculosis, we describe herein synthesis and biological evaluation of some substituted 4-arylthiazol-2-amino derivatives as modified analogues of the antiprotozoal drug Nitazoxanide (NTZ), which has recently been reported as potent inhibitor of Mtb H(37)Rv (Mtb MIC=52.12 μM) with an excellent ability to evade resistance. Among the synthesized derivatives, the two compounds 7a (MIC=15.28 μM) and 7c (MIC=17.03 μM) have exhibited about three times better Mtb growth inhibitory activity over NTZ and are free from any cytotoxicity (Vero CC(50) of 244 and 300 μM respectively). These two compounds represent promising leads for further optimization.     

Polymerization of Ftsz, a bacterial homolog of tubulin. is assembly cooperative?

FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis. In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers. Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or "isodesmic") assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration. We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme. The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates. It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolar K(D).     

Molecular docking studies of the interaction between propargylic enol ethers and human DNA topoisomerase IIα

Having identified a novel human DNA topoisomerase IIα (TOP2) catalytic inhibitor from a small and structure-focused library of propargylic enol ethers, we decided to analyze if the chirality of these compounds plays a determinant role in their antiproliferative activity. In this study, we describe for the first time the synthesis of the corresponding enantiomers and the biological evaluation against a panel of representative human solid tumor cell lines. Experimental results show that chirality does not influence the reported antiproliferative activity of these compounds. Docking studies of corresponding enantiomers against TOP2 reinforce the finding that the biological effect is not chiral-dependent and that these family of compounds seem to act as TOP2 catalytic inhibitors.