Biomedical subjects

Valakunja Nagaraja

Publications and source records attributed to Valakunja Nagaraja.

At least 19 recordsLinked to original sources

PubMed2007

Bacteriophage Mu C protein is a new member of unusual leucine zipper-HTH class of proteins.

Transcription activator protein C of bacteriophage Mu activates transcription of the late genes, including mom, during the lytic cycle of the phage. C binding to its site leads to the alteration in DNA topology of the promoter elements resulting in RNA polymerase (RNAP) recruitment. At the next step, the transactivator enhances promoter clearance of RNAP from P(mom). The C protein binds DNA with a very high affinity using a carboxyl-terminal helix turn helix (HTH) motif which has similarity with the HTH from paired domain of Drosophila prd protein. Previous studies established that the protein is dimeric in free and DNA bound forms. We describe now the unique dimerization interface of the protein. Two heptad repeats of hydrophobic amino acids found in the protein were considered to be the candidates for dimerization region. Site-directed mutational analysis revealed that the amino-terminal coiled coil region is not the dimerization determinant. In contrast, similar mutagenesis studies indicated a role for the leucine zipper motif, located in the middle region of the protein, in dimerization. Mixed oligomerization assays confirmed the importance of leucine zipper in C dimer formation establishing the presence of an uncommon zipper-HTH domain in the transactivator.

Amino Acid Sequence↗

PubMed2006

Glutamate racemase from Mycobacterium tuberculosis inhibits DNA gyrase by affecting its DNA-binding.

Glutamate racemase (MurI) catalyses the conversion of l-glutamate to d-glutamate, an important component of the bacterial cell wall. MurI from Escherichia coli inhibits DNA gyrase in presence of the peptidoglycan precursor. Amongst the two-glutamate racemases found in Bacillus subtilis, only one inhibits gyrase, in absence of the precursor. Mycobacterium tuberculosis has a single gene encoding glutamate racemase. Action of M.tuberculosis MurI on DNA gyrase activity has been examined and its mode of action elucidated. We demonstrate that mycobacterial MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species-specific as E.coli gyrase is also inhibited but is enzyme-specific as topoisomerase I activity remains unaltered. The mechanism of inhibition is different from other well-known gyrase inhibitors. MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA-binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalysed by DNA gyrase. MurI is thus not a typical potent inhibitor of DNA gyrase and instead its role could be in modulation of the gyrase activity.

Adenosine Triphosphatases↗

PubMed2006

Catalytic IgG from patients with hemophilia A inactivate therapeutic factor VIII.

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.

Adolescent↗

PubMed2006

A complex of DNA gyrase and RNA polymerase fosters transcription in Mycobacterium smegmatis.

We report here the existence of a complex between RNA polymerase (RNAP) and DNA gyrase in Mycobacterium smegmatis. The interaction between the two enzymes was detected during our attempts to purify DNA gyrase from M. smegmatis. RNAP subunits co-eluted along with DNA gyrase in two different affinity chromatography column procedures employed to purify the latter enzyme. A complex containing both the enzymes was isolated through gel filtration chromatography and sucrose density gradient centrifugation of the cell free extracts. The complex exhibited both DNA supercoiling and transcription activities. Reduction in the transcription activity of the complex in the presence of DNA gyrase inhibitor indicates a role for DNA gyrase in stimulating transcription.

Centrifugation, Density Gradient↗

PubMed2006

Indispensable, functionally complementing N and C-terminal domains constitute site-specific topoisomerase I.

Mycobacterium smegmatis topoisomerase I differs from the typical type IA topoisomerase in many properties. The enzyme recognizes both single and double-stranded DNA with high affinity and makes sequence-specific contacts during DNA relaxation reaction. The enzyme has a conserved N-terminal domain and a highly varied C-terminal domain, which lacks the characteristic zinc binding motifs found in most of the type I eubacterial enzymes. The roles of the individual domains of the enzyme in the topoisomerase I catalyzed reactions were examined by comparing the properties of full-length topoisomerase I with those of truncated polypeptides lacking the conserved N-terminal or the divergent C-terminal region. The N-terminal larger fragment retained the site-specific binding, DNA cleavage and religation properties, hallmark characteristics of the full-length M.smegmatis topoisomerase I. In contrast, the non-conserved C-terminal fragment lacking the typical DNA binding motif, exhibited non-specific DNA binding behaviour. The two polypeptide fragments, on their own do not catalyze DNA relaxation reaction. The relaxation activity is restored when both the fragments are mixed in vitro reconstituting the enzyme function. These results along with the DNA interaction pattern of the proteins implicate an essential role for the C-terminal region in single-strand DNA passage between the two transesterification reactions catalyzed by the N-terminal domain.

Bacterial Proteins↗

PubMed2006

Dual role for transactivator protein C in activation of mom promoter of bacteriophage Mu.

Transactivator C protein of bacteriophage Mu activates the mom gene of the phage by an unusual mechanism. DNA binding by C to its site results in unwinding of the neighboring sequences, realigning the out-of-phase promoter elements to facilitate RNA polymerase (RNAP) binding. High level stimulation of a C-independent constitutive promoter mutant (where RNAP is already bound) by the transactivator suggested an additional mechanism of transcription activation at a step after RNAP recruitment. In this study, we have investigated the various steps of promoter-polymerase interactions during transcription initiation by using both the promoter mutant and a positive control (pc) mutant of C protein. The transactivator does not influence formation of the open complex or its stability after facilitating the RNAP binding. However, at a subsequent step, the protein exerts an important role, enhancing the promoter clearance by increasing the productive RNAP.promoter complex. The pc mutant of the transactivator C is compromised at this step, supporting the additional downstream role for C in mom transcription activation. We suggest that this unusual multistep activation of Pmom has evolved to ensure irreversibility of the switch during the late lytic cycle of the phage.

Bacterial Proteins↗

PubMed2006

Physiopathology of catalytic antibodies: the case for factor VIII-hydrolyzing immunoglobulin G.

Antibodies that are able to catalyze the antigen for which they are specific are produced spontaneously by the immune system. Catalytic immunoglobulins (Igs) both of the IgM and IgG isotypes have been detected in the serum of healthy donors, where they have been proposed to participate in the removal of metabolic waste and in the defense of the organism against invading pathogens. Conversely, antigen-specific hydrolytic IgG have been reported in a number of inflammatory, autoimmune and neoplastic disorders: their pathogenic effects have been demonstrated occasionally. The pathophysiological relevance of catalytic antibodies thus remains an elusive issue. Through the description of the pro-coagulation factor VIII as a model target antigen for catalytic antibodies, we propose that catalytic antibodies have either a beneficial or a deleterious role depending on the physiopathological context. Physiology thus relies on a delicate equilibrium between the levels of soluble target antigen and that of antigen-specific hydrolyzing immunoglobulins. Indeed, in patients with hemophilia A, in whom endogenous factor VIII is deficient or missing and exogenous factor VIII needs to be administered to treat hemorrhagic events, the development of factor VIII-hydrolyzing IgG that inactivate the therapeutically administered factor VIII, may reveal deleterious. In contrast, in a situation in which excess factor VIII may be detrimental and lead to excessive coagulation, disseminated thrombosis and organ ischemia, as seen in severe sepsis, our recent data suggest that the presence of factor VIII-hydrolyzing IgG may be beneficial to the patient.

Antibodies, Catalytic↗

PubMed2006

Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase.

KpnI DNA-(N6-adenine) methyltransferase (M.KpnI) recognises the sequence 5'-GGTACC-3' and transfers the methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.KpnI exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.KpnI was obtained based on protein fold-recognition analysis, using the crystal structures of M.RsrI and M.MboIIA as templates. Residues I146, I161 and Y167, the side chains of which are present in the putative dimerisation interface in the model, were targeted for site-directed mutagenesis. Methylation and in vitro restriction assays showed that the mutant MTases are catalytically inactive. Mutation at the I146 position resulted in complete disruption of the dimer. The replacement of I146 led to drastically reduced DNA and cofactor binding. Substitution of I161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.KpnI to catalyse the methyl transfer reaction.

Amino Acid Sequence↗

PubMed2006

Evaluation of antimycobacterial and DNA gyrase inhibition of fluoroquinolone derivatives.

The antimycobacterial activity (both in vitro and in vivo) and DNA gyrase inhibition of newly synthesized fluoroquinolone derivatives were tested against Mycobacterium tuberculosis H(37)Rv and Mycobacterium smegmatis, respectively. Among the synthesized compounds, compound F11 was found to exhibit the most potent in vitro antimycobacterial activity with a MIC value of 0.78 microg/ml, and a selectivity index of more than 80 while not being cytotoxic to the Vero cell line up to 62.5 microg/ml. When evaluated for in vivo antimycobacterial activity, compound F11 demonstrated a paramount decrease of bacterial load in lung and spleen tissues compared to the control and better than the standard drug ciprofloxacin.

Animals↗

PubMed2005

Pathophysiology of catalytic antibodies.

Immunoglobulins have initially been illustrated as proteins produced by the immune system for binding and neutralizing foreign molecules potentially harmful to the organism. The number of V(H), D(H), J(H), V(L) and J(L) genes that encode the variable regions of immunoglobulins and the junctional diversity that occurs at the time of somatic rearrangement determine the extent of the repertoire of antibodies that may be potentially produced by an organism. This potential repertoire includes antibodies the antigen binding site of which may recognize external as well as autologous antigens, or may structurally resemble the active site of enzymes and be endowed with enzymatic activity. Under physiological conditions, B cell clones that produce antibodies naturally endowed with catalytic activity are negatively regulated and subjected to apoptosis. Catalytic antibodies are expressed only following active immunization, or if the physiological regulatory mechanisms that control the expression of catalytic antibody-producing B cell clones are perturbed, e.g. in the context of pregnancy or in the course of autoimmune diseases.

Antibodies, Catalytic↗

PubMed2005

Synthesis and antimycobacterial evaluation of various 7-substituted ciprofloxacin derivatives.

Tuberculosis continues to be a major cause of morbidity and mortality all over the world. Various 7-substituted ciprofloxacin derivatives were synthesized and evaluated for antimycobacterial activity in vitro and in vivo against Mycobacterium tuberculosis and for inhibition of the supercoiling activity of DNA gyrase from Mycobacterium smegmatis. Preliminary results indicated that most of the compounds demonstrated better in vitro antimycobacterial activity against M. tuberculosis than ciprofloxacin. Compound 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-[[N4-[1'-(5-methylisatinyl-beta-semicarbazo)]methyl]N1-piperazinyl]-3-quinoline carboxylic acid (3h) decreased the bacterial load in spleen tissue with 0.76-log10 protections and was considered to be moderately active in reducing bacterial count in spleen. The results demonstrated the potential and importance of developing new quinolone derivatives against mycobacterial infections.

Animals↗

PubMed2005

A monoclonal antibody that inhibits mycobacterial DNA gyrase by a novel mechanism.

DNA gyrase is a DNA topoisomerase indispensable for cellular functions in bacteria. We describe a novel, hitherto unknown, mechanism of specific inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis DNA gyrase by a monoclonal antibody (mAb). Binding of the mAb did not affect either GyrA-GyrB or gyrase-DNA interactions. More importantly, the ternary complex of gyrase-DNA-mAb retained the ATPase activity of the enzyme and was competent to catalyse DNA cleavage-religation reactions, implying a new mode of action different from other classes of gyrase inhibitors. DNA gyrase purified from fluoroquinolone-resistant strains of M.tuberculosis and M.smegmatis were inhibited by the mAb. The absence of cross-resistance of the drug-resistant enzymes from two different sources to the antibody-mediated inhibition corroborates the new mechanism of inhibition. We suggest that binding of the mAb in the proximity of the primary dimer interface region of GyrA in the heterotetrameric enzyme appears to block the release of the transported segment after strand passage, leading to enzyme inhibition. The specific inhibition of mycobacterial DNA gyrase with the mAb opens up new avenues for designing novel lead molecules for drug discovery and for probing gyrase mechanism.

Adenosine Triphosphatases↗

PubMed2005

High levels of catalytic antibodies correlate with favorable outcome in sepsis.

Sepsis is the leading cause of death in intensive care units and results from a deleterious systemic host response to infection. Although initially perceived as potentially deleterious, catalytic antibodies have been proposed to participate in removal of metabolic wastes and protection against infection. Here we show that the presence in plasma of IgG endowed with serine protease-like hydrolytic activity strongly correlates with survival from sepsis. Variances of catalytic rates of IgG were greater in the case of patients with severe sepsis than healthy donors (P < 0.001), indicating that sepsis is associated with alterations in plasma levels of hydrolytic IgG. The catalytic rates of IgG from patients who survived were significantly greater than those of IgG from deceased patients (P < 0.05). The cumulative rate of survival was higher among patients exhibiting high rates of IgG-mediated hydrolysis as compared with patients with low hydrolytic rates (P < 0.05). An inverse correlation was also observed between the markers of severity of disseminated intravascular coagulation and rates of hydrolysis of patients' IgG. Furthermore, IgG from three surviving patients hydrolyzed factor VIII, one of which also hydrolyzed factor IX, suggesting that, in some patients, catalytic IgG may participate in the control of disseminated microvascular thrombosis. Our observations provide the first evidence that hydrolytic antibodies might play a role in recovery from a disease.

Antibodies, Catalytic↗

PubMed2005

An atypical type II topoisomerase from Mycobacterium smegmatis with positive supercoiling activity.

Topoisomerases are essential ubiquitous enzymes, falling into two distinct classes. A number of eubacteria including Escherichia coli, typically contain four topoisomerases, two type I topoisomerases and two type II topoisomerases viz. DNA gyrase and topoisomerase IV. In contrast several other bacterial genomes including mycobacteria, encode for one type I topoisomerase and a DNA gyrase. Here we describe a new type II topoisomerase from Mycobacterium smegmatis which is different from DNA gyrase or topoisomerase IV in its characteristics and origin. The topoisomerase is distinct with respect to domain organization, properties and drug sensitivity. The enzyme catalyses relaxation of negatively supercoiled DNA in an ATP-dependent manner and also introduces positive supercoils to both relaxed and negatively supercoiled substrates. The genes for this additional topoisomerase are not found in other sequenced mycobacterial genomes and may represent a distant lineage.

Ciprofloxacin↗

PubMed2004

Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily.

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5'-GGTAC--C-3' in the presence of Mg2+ as shown generating 3' four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a betabetaalpha-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD...D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.

Amino Acid Sequence↗

PubMed2004

Ca(2+)-mediated site-specific DNA cleavage and suppression of promiscuous activity of KpnI restriction endonuclease.

The characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg(2+) requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca(2+) ions, comparable with that of Mg(2+), is previously not described. Most intriguingly, KpnI REase exhibits Ca(2+)-dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn(2+) or Mg(2+), with the complete suppression of promiscuous activity in the presence of Ca(2+). KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease.

Calcium↗

PubMed2004

KpnI restriction endonuclease and methyltransferase exhibit contrasting mode of sequence recognition.

The molecular basis of the interaction of KpnI restriction endonuclease (REase) and the corresponding methyltransferase (MTase) at their cognate recognition sequence is investigated using a range of footprinting techniques. DNase I protection analysis with the REase reveals the protection of a 14-18 bp region encompassing the hexanucleotide recognition sequence. The MTase, in contrast, protects a larger region. KpnI REase contacts two adjacent guanine residues and the single adenine residue in both the strands within the recognition sequence 5'-GGTACC-3', inferred by dimethylsulfate (DMS) protection, interference and missing nucleotide interference analysis. In contrast, KpnI MTase does not show elaborate base-specific contacts. Ethylation interference analysis also showed the differential interaction of REase and MTase with phosphate groups of three adjacent bases on both strands within the recognition sequence. The single thymine residue within the sequence is hyper- reactive to the permanganate oxidation, consistent with MTase-induced base flipping. The REase on the other hand does not show any major DNA distortion. The results demonstrate that the differences in the molecular interaction pattern of the two proteins at the same recognition sequence reflect the contrasting chemistry of DNA cleavage and methylation catalyzed by these two dissimilar enzymes, working in combination as constituents of a cellular defense strategy.

Base Sequence↗

PubMed2003

Chromosomally encoded gyrase inhibitor GyrI protects Escherichia coli against DNA-damaging agents.

DNA gyrase, a type II topoisomerase, is the sole supercoiling activity in the cell and is essential for cell survival. There are two proteinaceous inhibitors of DNA gyrase that are plasmid-borne and ensure maintenance of the plasmids in bacterial populations. However, the physiological role of GyrI, an inhibitor of DNA gyrase encoded by the Escherichia coli genome, has been elusive. Previously, we have shown that GyrI imparts resistance against microcin B17 and CcdB. Here, we find that GyrI provided partial/limited protection against the quinolone class of gyrase inhibitors but had no effect on inhibitors that interfere with the ATPase activity of the enzyme. Moreover, GyrI negated the effect of alkylating agents, such as mitomycin C and N-methyl- N-nitro- N-nitrosoguanidine, that act independently of DNA gyrase. Hence, in vivo, GyrI appears to be involved in reducing DNA damage from many sources. In contrast, GyrI is not effective against lesions induced by ultraviolet radiation. Furthermore, the expression of GyrI does not significantly alter the topology of DNA. Thus, although isolated as an inhibitor of DNA gyrase, GyrI seems to have a broader role in vivo than previously envisaged.

Alkylating Agents↗