Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (Mstopol) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage m the absence of Mg2+ but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim. (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+, in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike Mstopol and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.

Development and characterization of lysine based tripeptide analogues as inhibitors of Sir2 activity

Sirtuins are NAD(+) dependent deacetylases that modulate various essential cellular functions. Development of peptide based inhibitors of Sir2s would prove useful both as pharmaceutical agents and as effectors by which downstream cellular alterations can be monitored. Click chemistry that utilizes Huisgen's 1,3-dipolar cycloaddition permits attachment of novel modifications onto the side chain of lysine. Herein, we report the synthesis of peptide analogues prepared using click reactions on N epsilon-propargyloxycarbonyl protected lysine residues and their characterization as inhibitors of Plasmodium falciparum Sir2 activity. The peptide based inhibitors exhibited parabolic competitive inhibition with respect to acetylated-peptide substrate and parabolic non-competitive inhibition with NAD(+) supporting the formation of EI2 and E.NAD(+).I-2 complexes. Cross-competition inhibition analysis with the non-competitive inhibitor nicotinamide (NAM) ruled out the possibility of the NAM-binding site being the second inhibitor binding site, suggesting the presence of a unique alternate pocket commodating the inhibitor. One of these compounds was also found to be a potent inhibitor of the intraerythrocytic growth of P. falciparum with 50% inhibitory concentration in the micromolar range.

Pi-turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence.

The i + 5-->i hydrogen bonded turn conformation (pi-turn) with the fifth residue adopting alpha L conformation is frequently found at the C-terminus of helices in proteins and hence is speculated to be a "helix termination signal." An analysis of the occurrence of i + 5-->i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C-terminus), using coordinates of 228 protein crystal structures determined by X-ray crystallography to better than 2.5 A resolution is reported in this paper. Of 486 detected pi-turn conformations, 367 have the (i + 4)th residue in alpha L conformation, generally occurring at the C-terminus of alpha-helices, consistent with previous observations. However, a significant number (111) of pi-turn conformations occur with (i + 4)th residue in alpha R conformation also, generally occurring in alpha-helices as distortions either at the terminii or at the middle, a novel finding. These two sets of pi-turn conformations are referred to by the names pi alpha L and pi alpha R-turns, respectively, depending upon whether the (i + 4)th residue adopts alpha L or alpha R conformations. Four pi-turns, named pi alpha L'-turns, were noticed to be mirror images of pi alpha L-turns, and four more pi-turns, which have the (i + 4)th residue in beta conformation and denoted as pi beta-turns, occur as a part of hairpin bend connecting twisted beta-strands. Consecutive pi-turns occur, but only with pi alpha R-turns. The preference for amino acid residues is different in pi alpha L and pi alpha R-turns. However, both show a preference for Pro after the C-termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of pi alpha L-turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in pi alpha L-turns is mainly Gly and less often Asn. Although pi alpha R-turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for pi alpha R-turns, whereas good helix former Ala is not preferred. pi-Turns in peptides provide a picture of the pi-turn at atomic resolution. Only nine peptide-based pi-turns are reported so far, and all of them belong to pi alpha L-turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.

Ligand dependent intra and inter subunit communication in human tryptophanyl tRNA synthetase as deduced from the dynamics of structure networks

Homodimeric protein tryptophanyl tRNA synthetase (TrpRS) has a Rossmann fold domain and belongs to the 1c subclass of aminoacyl tRNA synthetases. This enzyme performs the function of acylating the cognate tRNA. This process involves a number of molecules (2 protein subunits, 2 tRNAs and 2 activated Trps) and thus it is difficult to follow the complex steps in this process. Structures of human TrpRS complexed with certain ligands are available. Based on structural and biochemical data, mechanism of activation of Trp has been speculated. However, no structure has yet been solved in the presence of both the tRNA(Trp) and the activated Trp (TrpAMP). In this study, we have modeled the structure of human TrpRS bound to the activated ligand and the cognate tRNA. In addition, we have performed molecular dynamics (MD) simulations on these models as well as other complexes to capture the dynamical process of ligand induced conformational changes. We have analyzed both the local and global changes in the protein conformation from the protein structure network (PSN) of MD snapshots, by a method which was recently developed in our laboratory in the context of the functionally monomeric protein, methionyl tRNA synthetase. From these investigations, we obtain important information such as the ligand induced correlation between different residues of this protein, asymmetric binding of the ligands to the two subunits of the protein as seen in the crystal structure analysis, and the path of communication between the anticodon region and the aminoacylation site. Here we are able to elucidate the role of dimer interface at a level of detail, which has not been captured so far.

Titration calorimetric studies to elucidate the specificity of the interactions of polymyxin B with lipopolysaccharides and lipid A

Lipopolysaccharide (LPS), the major cell wall constituent of Gram-negative bacteria, evokes a multitude of biological effects in mammals including pyrogenicity and toxic shock syndrome. Polymyxin B (PmB), a polycationic cyclic peptide, is known to neutralize most of its activities. The nature of the interaction of PmB with LPS and lipid A was investigated by isothermal titration calorimetry. PmB binds to LPS as well as lipid A stoichiometrically and non-co-operatively with micromolar affinity. These interactions are driven primarily by a favourable change in entropy (delta S) and are endothermic in nature. These positive changes in enthalpies decrease with increasing temperature, yielding a heat capacity change, delta Cp, of -2385 J.mol-1.degree-1 for PmB-LPS interactions while the binding of PmB to lipid A displays a delta Cp of -2259 J.mol-1.degree-1. The negative heat capacity changes provide strong evidence for the role of hydrophobic interactions as the driving force for the association of PmB with LPS and lipid A. A correlation of the energetics of these interactions with analyses of the molecular models of PmB suggests that a cluster of solvent-exposed non-polar amino acid side-chains that line one surface of the molecule, together with a ring of positively charged residues on its other surface, are responsible for its strong and stoichiometric binding to LPS.

Functional characterization of the precursor and spliced forms of RecA protein of Mycobacterium tuberculosis

The recA locus of pathogenic mycobacteria differs from that of nonpathogenic species because it contains large intervening sequences nested in the RecA homology region that are excised by an unusual protein-splicing reaction. In vivo assays indicated that Mycobacterium tuberculosis recA partially complemented Escherichia coli recA mutants for recombination and mutagenesis. Further, splicing of the 85 kDa precursor to 38 kDa MtRecA protein was necessary for the display of its activity, in vivo. To gain insights into the molecular basis for partial and lack of complementation by MtRecA and 85 kDa proteins, respectively, we purified both of them to homogeneity. MtRecA protein, but not the 85 kDa form, bound stoichiometrically to single-stranded DNA in the presence of ATP. MtRecA protein was cross-linked to 8-azidoadenosine 5'-triphosphate with reduced efficiency, and kinetic analysis of ATPase activity suggested that it is due to decreased affinity for ATP. In contrast, the 85 kDa form was unable to bind ATP, in the presence or absence of ssDNA and, consequently, was entirely devoid of ATPase activity. Molecular modeling studies suggested that the decreased affinity of MtRecA protein for ATP and the reduced efficiency of its hydrolysis might be due to the widening of the cleft which alters the hydrogen bonds and the contact area between the enzyme and the substrate and changes in the disposition of the amino acid residues around the magnesium ion and the gamma-phosphate. The formation of joint molecules promoted by MtRecA protein was stimulated by SSB when the former was added first. The probability of an association between the lack and partial levels of biological activity of RecA protein(s) to that of illegitimate recombination in pathogenic mycobacteria is considered.

Impacts of agriculture on amphibians at multiple scales

Agriculture-mediated habitat loss and degradation together with climate change are among the greatest global threats to species, communities, and ecosystem functioning. During the last century, more than 50% of the world's wetlands have been lost and agricultural activities have subjected wetland species to increased isolation and decreased quality of habitats. Likewise, as a part of agricultural intensification, the use of pesticides has increased notably, and pesticide residues occur frequently in wetlands making the exposure of wetland organisms to pesticides highly probable. In this thesis, a set of ecotoxicological and landscape ecological studies were carried out to investigate pesticide-effects on tadpoles, and species-habitat relationships of amphibians in agricultural landscapes. The results show that the fitness of R. temporaria tadpoles can be negatively affected by sublethal pesticide concentrations, and that pesticides may increase the costs of response to natural environmental stressors. However, tadpoles may also be able to compensate for some of the negative effects of pesticides. The results further demonstrate that both historic and current-day agricultural land use can negatively impact amphibians, but that in some cases the costs of living in agricultural habitats may only become apparent when amphibians face other environmental stressors, such as drought. Habitat heterogeneity may, however, increase the persistence of amphibians in agricultural landscapes. Hence, the results suggest that amphibians are likely to be affected by agricultural processes that operate at several spatial and temporal scales, and that it is probable that various processes related to current-day agriculture will affect both larval and adult amphibians. The results imply that maintaining dense wetland patterns could enhance persistence of amphibian populations in agricultural habitats, and indicate that heterogeneous landscapes may lower the risk of regional amphibian population declines under extreme weather perturbations.

Structure and Dynamics of Coil-like Molecules by Residual Dipolar Couplings

Nuclear magnetic resonance (NMR) spectroscopy provides us with many means to study biological macromolecules in solution. Proteins in particular are the most intriguing targets for NMR studies. Protein functions are usually ascribed to specific three-dimensional structures but more recently tails, long loops and non-structural polypeptides have also been shown to be biologically active. Examples include prions, -synuclein, amylin and the NEF HIV-protein. However, conformational preferences in coil-like molecules are difficult to study by traditional methods. Residual dipolar couplings (RDCs) have opened up new opportunities; however their analysis is not trivial. Here we show how to interpret RDCs from these weakly structured molecules. The most notable residual dipolar couplings arise from steric obstruction effects. In dilute liquid crystalline media as well as in anisotropic gels polypeptides encounter nematogens. The shape of a polypeptide conformation limits the encounter with the nematogen. The most elongated conformations may come closest whereas the most compact remain furthest away. As a result there is slightly more room in the solution for the extended than for the compact conformations. This conformation-dependent concentration effect leads to a bias in the measured data. The measured values are not arithmetic averages but essentially weighted averages over conformations. The overall effect can be calculated for random flight chains and simulated for more realistic molecular models. Earlier there was an implicit thought that weakly structured or non-structural molecules would not yield to any observable residual dipolar couplings. However, in the pioneering study by Shortle and Ackerman RDCs were clearly observed. We repeated the study for urea-denatured protein at high temperature and also observed indisputably RDCs. This was very convincing to us but we could not possibly accept the proposed reason for the non-zero RDCs, namely that there would be some residual structure left in the protein that to our understanding was fully denatured. We proceeded to gain understanding via simulations and elementary experiments. In measurements we used simple homopolymers with only two labelled residues and we simulated the data to learn more about the origin of RDCs. We realized that RDCs depend on the position of the residue as well as on the length of the polypeptide. Investigations resulted in a theoretical model for RDCs from coil-like molecules. Later we extended the studies by molecular dynamics. Somewhat surprisingly the effects are small for non-structured molecules whereas the bias may be large for a small compact protein. All in all the work gave clear and unambiguous results on how to interpret RDCs as structural and dynamic parameters of weakly structured proteins.

NUVIEW:software for display and interactive manipulation of nucleic acid models

The NUVIEW software package allows skeletal models of any double helical nucleic acid molecule to be displayed out a graphics monitor and to apply various rotations, translations and scaling transformations interactively, through the keyboard. The skeletal model is generated by connecting any pair of representative points, one from each of the bases in the basepair. In addition to the above mentioned manipulations, the base residues can be identified by using a locator and the distance between any pair of residues can be obtained. A sequence based color coded display allows easy identification of sequence repeats, such as runs of Adenines. The real time interactive manipulation of such skeletal models for large DNA/RNA double helices, can be used to trace the path of the nucleic acid chain in three dimensions and hence get a better idea of its topology, location of linear or curved regions, distances between far off regions in the sequence etc. A physical picture of these features will assist in understanding the relationship between base sequence, structure and biological function in nucleic acids.

Identification of alpha- and beta-Hydroxy Acid Containing Cyclodepsipeptides in Natural Peptide Mixtures Using Negative Ion Mass Spectrometry

Natural peptide libraries often contain cyclodepsipeptides containing alpha or beta hydroxy residues. Extracts of fungal hyphae of Isaria yield a microheterogenous cyclodepsipeptide mixture in which two classes of molecules can be identified by mass spectral fragmentation of negative ions. In the case of isaridins, which contain an alpha-hydroxy residue and a beta-amino acid residue, a characteristic product ion corresponding to a neutral loss of 72 Da is obtained. hi addition, neutral loss of water followed by a 72 Da loss is also observed. Two distinct modes of fragmentation rationalize the observed product ion distribution. The neutral loss of 72 Da has also been obtained for a roseotoxin component, which is also an alpha-hydroxy residue containing cyclodepsipeptide. In the case of isariins, which contain a beta-hydroxy acid residue, ring opening and subsequent loss of the terminal residue as an unsaturated ketene fragment, rationalizes the observed product ion formation. Fragmentation of negative ions provide characteristic neutral losses, which are diagnostic of the presence of alpha-hydroxy or beta-hydroxy residues.

Methylenetetrahydrofolate reductase CpG islands: Epigenotyping

Background Determination of the differential DNA methylation patterns of methylenetetrahydrofolate reductase (MTHFR) that are associated with differential MTHFR activity is important to understand the pathogenesis of ischemic stroke. However, to date, no data are available on the differential DNA methylation profiles of Kelantanese Malays. Therefore, we developed a rapid and efficient serial pyrosequencing assay to determine differential DNA methylation profiles of MTHFR, which help to further our understanding of the pathogenesis of ischemic stroke. The developed assay also served as the validation platform for our previous computational epigenetic research on MTHFR. Methods Polymerase chain reaction primers were designed and validated to specifically amplify the cytosine that is followed by guanine residues (CpGs) A and B regions. Prior epigenotyping on 110 Kelantanese Malays, the serial pyrosequencing assays for the CpGs A and B regions were validated using five validation controls. The mean values of the DNA methylation profiles of CpGs A and B were calculated. Results The mean DNA methylation levels for CpGs A and B were 0.984 ± 0.582 and 2.456 ± 1.406, respectively. The CpGs 8 and 20 showed the highest (5.581 ± 4.497) and the lowest (0.414 ± 2.814) levels of DNA methylation at a single-base resolution. Conclusion We have successfully developed and validated a pyrosequencing assay that is fast and can yield high-quality pyrograms for DNA methylation analysis and is therefore applicable to high throughput study. Using this newly developed pyrosequencing assay, the MTHFR DNA methylation profiles of 110 Kelantanese Malays were successfully determined. It also validated our computational epigenetic research on MTHFR.

Identification and Characterization of a Novel Deoxyhypusine Synthase in Leishmania donovani

Deoxyhypusine synthase, an NAD(+)-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (N-epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.

Mycobacterium tuberculosis FtsZ requires at least one arginine residue at the C-terminal end for polymerization in vitro

We examined whether C-terminal residues of soluble recombinant FtsZ of Mycobacterium tuberculosis (MtFtsZ) have any role in MtFtsZ polymerization in vitro. MtFtsZ-delta C1, which lacks C-terminal extreme Arg residue (underlined in the C-terminal extreme stretch of 13 residues, DDDDVDVPPFMRR), but retaining the penultimate Arg residue (DDDDVDVPPFMR), polymerizes like full-length MtFtsZ in vitro. However, MtFtsZ-delta C2 that lacks both the Arg residues at the C-terminus (DDDDVDVPPFM), neither polymerizes at pH 6.5 nor forms even single- or double-stranded filaments at pH 7.7 in the presence of 10 mM CaCl2. Neither replacement of the penultimate Arg residue, in the C-terminal Arg deletion mutant DDDDVDVPPFMR, with Lys or His or Ala or Asp (DDDDVDVPPFMK/H/A/D) enabled polymerization. Although MtFtsZ-delta C2 showed secondary and tertiary structural changes, which might have affected polymerization, GTPase activity of MtFtsZ-delta C2 was comparable to that of MtFtsZ. These data suggest that MtFtsZ requires an Arg residue as the extreme C-terminal residue for polymerization in vitro. The polypeptide segment containing C-terminal 67 residues, whose coordinates were absent from MtFtsZ crystal structure, was modeled on tubulin and MtFtsZ dimers. Possibilities for the influence of the C-terminal Arg residues on the stability of the dimer and thereby on MtFtsZ polymerization have been discussed.

Ligand Specificity of Group I Biotin Protein Ligase of Mycobacterium tuberculosis

Background: Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC. Methodology/Principal Findings: BPL/BirA (Biotin Protein Ligase), and its substrate, biotin carboxyl carrier protein (BCCP) of Mycobacterium tuberculosis (Mt) were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the similar to 29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5'AMP liganded forms. This was confirmed by molecular weigt profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS). Computational docking of biotin and bio-5'AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5'AMP. Docking simulations also suggest that bio-5'AMP hydrogen bonds to the conserved `GRGRRG' sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The K-m for BCCP was similar to 5.2 mu M and similar to 420 nM for biotin. MtBPL has low affinity (K-b = 1.06 x 10(-6) M) for biotin relative to EcBirA but their K-m are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5'AMP by EcBirA is channeled for its repressor activity. Conclusions/Significance: These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.

Synthetic arabinomannan glycolipids and their effects on growth and motility of the Mycobacterium smegmatis

Arabinomannan-containing glycolipids, relevant to the mycobacterial cell-wall component lipoarabinomannan, were synthesized by chemical methods. The glycolipids were presented with tri- and tetrasaccharide arabinomannans as the sugar portion and a double alkyl chain as the lyophilic portion. Following synthesis, systematic biological and biophysical studies were undertaken in order to identify the effects of the glycolipids during mycobacterium growth. The studies included mycobacterial growth, biofilm formation and motility assays. From the studies, it was observed that the synthetic glycolipid with higher arabinan residues inhibited the mycobacterial growth, lessened the biofilm formation and impaired the motility of mycobacteria. A surface plasmon resonance study involving the immobilized glycan surface and the mycobacterial crude lysates as analytes showed specificities of the interactions. Further, it was found that cell lysates from motile bacteria bound oligosaccharide with higher affinity than non-motile bacteria.