Rama Voladri - Academia.edu (original) (raw)

Papers by Rama Voladri

Research paper thumbnail of Content Alerts

This article cites 22 articles, 8 of which can be accessed free at:

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Research paper thumbnail of Ketoreductase polypeptides for producing azetidinone

A polypeptide ketoreductase capable of converting the substrate, methyl 2-benzamidomethyl-3-oxobu... more A polypeptide ketoreductase capable of converting the substrate, methyl 2-benzamidomethyl-3-oxobutyrate, the product, 2S, 3R-methyl-2-benzamidomethyl-3-hydroxybutyrate, with an estereomerico excess percentage of at least 60%, comprising an amino acid sequence: (i) that is at least 85% identical to the reference sequence based on the sEQUENTIAL IDENTIFICATION NUMBER: 2, 4 or 86 having the following characteristics: the corresponding X 94 residue is threonine; the residue corresponding to X 199 is histidine and residue corresponding to X 202 is valine or leucine; and (ii) in which the residue corresponding to X 94 is alanine or threonine; the residue corresponding to X 199 is alanine, histidine or asparagine; and residue corresponding to X 202 is valine or leucine.

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Research paper thumbnail of Variants de cellobiohydrolase

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Research paper thumbnail of Biocatalyseurs et Procédés D'Hydroxylation De Composés Chimiques

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Research paper thumbnail of Enhanced production of recombinant Mycobacterium tuberculosis antigen 85B in Salmonella by replacement of low-usage codons

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Research paper thumbnail of Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model

Infection and Immunity, 1997

Isogeneic bacterial strains that differ only in the production of a single microbial factor have ... more Isogeneic bacterial strains that differ only in the production of a single microbial factor have been invaluable in studying the pathogenesis of bacterial infections. The targeted, intentional inactivation of a gene encoding a potential virulence determinant generally requires homologous recombination to replace the gene with an inactivated allele. To determine whether the insertion and expression of a fragment of a bacterial gene in an antisense orientation could be used as a rapid alternative to allelic inactivation for producing paired isogeneic isolates, we inverted a 600-bp fragment of the Staphylococcus aureus gene encoding alpha-toxin, hla, behind its native promoter on an Escherichia coli-S. aureus shuttle vector. A transformant of an S. aureus strain carrying the antisense hla fragment produced antisense hla RNA and made 16-fold less alpha-toxin than either its parent or an isogeneic transformant containing vector DNA without hla. Also, intraperitoneal injection of 1.5 x 10...

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Research paper thumbnail of Characterization of a Chromosomal Gene Encoding Type B β-Lactamase in Phage Group II Isolates ofStaphylococcus aureus

Antimicrobial Agents and Chemotherapy, 1998

In contrast to most Staphylococcus aureus isolates in which the gene for staphylococcal β-lactama... more In contrast to most Staphylococcus aureus isolates in which the gene for staphylococcal β-lactamase (blaZ) is plasmid borne, isolates typeable by group II bacteriophages frequently carry blaZ on the chromosome. Furthermore, the chromosomal gene encodes the type B variant of staphylococcal β-lactamase for which the nucleotide and deduced amino acid sequences have not yet been reported. To better understand β-lactamase production among phage group II staphylococci and the nature of the type B β-lactamase, we determined the type and amount of enzyme produced by 24 phage group II isolates. Of these isolates, 1 did not produce β-lactamase, 8 produced the type B enzyme, and 15 produced the type C enzyme. In all eight type B β-lactamase-producing isolates, blaZ was located on the chromosome. This was in contrast to the type C β-lactamase-producing isolates, in which blaZ was located on a 21-kb plasmid. The nucleotide sequence corresponding to the leader peptide and the N-terminal 85% of th...

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Research paper thumbnail of Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216

Journal of Bacteriology, 1996

beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lac... more beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of Staphylococcus aureus beta-lactamase, designated A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in primary structure by only a few amino acids. Using the reported sequences of the A, C, and D enzymes along with crystallographic data about the structure of the type A enzyme to identify amino acid differences located close to the active site, we hypothesized that these differences might explain the kinetic heterogeneity of the wild-type beta-lactamases. To test this hypothesis, genes encoding the type A, C, and D beta-lactamases were modified by site-directed mutagenesis, yielding mutant enzymes with single amino acid substitutions. The substitution of asparagine for serine at residue 216 of type A beta-lactamase resulted in a kinetic profi...

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Research paper thumbnail of Improved Ketoreductase Polypeptides for the Stereoselective Production of (R)-3-HYDROXYTHIOLANE

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Research paper thumbnail of Polynucleotides Encoding Engineered Ketoreductase Polypeptides

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Research paper thumbnail of Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane

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Research paper thumbnail of Ketoreductase polypeptides and uses thereof

The present disclosure provides ketoreductase enzymes that have been engineered with wild-type ke... more The present disclosure provides ketoreductase enzymes that have been engineered with wild-type ketoreductase enzyme and improved properties compared to naturally occurring. Polynucleotides encoding the engineered ketoreductase enzyme, the host cell capable of expressing the engineered ketoreductase enzyme, and methods of synthesizing the various chiral compounds using the engineered ketoreductase enzyme also provided. In one aspect, the present disclosure is 2- [3- [3- [2- (7-chloro-2-quinolinyl) ethenyl] phenyl] -3-oxopropyl] benzoic acid methyl ester ( "substrate") ( S, E) - can be reduced to methyl 2- (3- (3- (2- (7-chloro-2-yl) vinyl) phenyl) -3-hydroxypropyl) benzoate ( "product") or to provide a ketoreductase polypeptide capable of converting.

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Research paper thumbnail of Ketoreductase Polypeptides for the Production of Azetidinone

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Research paper thumbnail of Highly Enantioselective Reduction of a Small Heterocyclic Ketone: Biocatalytic Reduction of Tetrahydrothiophene-3-one to the Corresponding (R)-Alcohol

Organic Process Research & Development, 2010

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Research paper thumbnail of Highly Enantioselective Reduction of a Small Heterocyclic Ketone: Biocatalytic Reduction of Tetrahydrothiophene-3-one to the Corresponding (R)-Alcohol

Organic Process Research & Development, 2010

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Research paper thumbnail of Development of a Practical Biocatalytic Process for ( R )-2-Methylpentanol

Organic Process Research & Development, 2010

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Research paper thumbnail of Growth of Staphylococcus aureus with Nafcillin In Vitro Induces  -Toxin Production and Increases the Lethal Activity of Sterile Broth Filtrates in a Murine Model

Journal of Infectious Diseases, 1995

The morbidity and mortality of Staphylococcus aureus infections remain high despite antibiotic th... more The morbidity and mortality of Staphylococcus aureus infections remain high despite antibiotic therapy. To investigate further the observation that penicillins increase the hemolytic activity of staphylococcal cultures, 37 strains were grown in broth with and without subinhibitory nafcillin. Nafcillin stimulated hemolytic activity in nafcillin-susceptible and -resistant isolates. Sterile broth filtrates of nafcillin-associated cultures injected intraperitoneally in mice were more rapidly lethal than filtrates of the same strain grown without nafcillin. Lethality was neutralized by anti-alpha-toxin antisera. DNA-RNA hybridization revealed a nafcillin-associated increase in alpha-toxin mRNA during the postexponential growth phase after the activation of agr. Isolates grown in slightly inhibitory nafcillin concentrations had more alpha-toxin mRNA than did nafcillin-free cultures, whereas agr RNAIII levels were comparable. This suggests that nafcillin-induced alpha-toxin production is not entirely attributable to agr. A supplemental regulatory mechanism may be involved.

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Research paper thumbnail of β‐Lactamase Production Diminishes the Prophylactic Efficacy of Ampicillin and Cefazolin in a Guinea Pig Model of Staphylococcus aureus Wound Infection

The Journal of Infectious Diseases, 1998

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Research paper thumbnail of Enhanced Production of Recombinant Mycobacterium tuberculosis Antigens in Escherichia coli by Replacement of Low-Usage Codons

Infection and Immunity, 2000

A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is t... more A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is the inability to produce large quantities of these proteins. To test the hypothesis that poor expression of some mycobacterial genes in Escherichia coli is due, in part, to the presence of low-usage E. coli codons, we used site-directed mutagenesis to convert low-usage codons to high-usage codons for the same amino acid in the Mycobacterium tuberculosis genes for antigens 85A and 85B and superoxide dismutase. Replacement of five codons in the wild-type gene for antigen 85B increased recombinant protein production in E. coli 54-fold. The recombinant antigen elicited proliferation and gamma interferon production by lymphocytes from healthy tuberculin reactors and was recognized by monoclonal antibodies to native antigen 85, indicating that the recombinant antigen contained T-cell and B-cell epitopes. Northern blotting demonstrated only a 1.7- to 2.5-fold increase in antigen 85B mRNA, sugges...

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Research paper thumbnail of Iron-cofactored Superoxide Dismutase Inhibits Host Responses toMycobacterium tuberculosis

American Journal of Respiratory and Critical Care Medicine, 2001

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Research paper thumbnail of Content Alerts

This article cites 22 articles, 8 of which can be accessed free at:

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Research paper thumbnail of Ketoreductase polypeptides for producing azetidinone

A polypeptide ketoreductase capable of converting the substrate, methyl 2-benzamidomethyl-3-oxobu... more A polypeptide ketoreductase capable of converting the substrate, methyl 2-benzamidomethyl-3-oxobutyrate, the product, 2S, 3R-methyl-2-benzamidomethyl-3-hydroxybutyrate, with an estereomerico excess percentage of at least 60%, comprising an amino acid sequence: (i) that is at least 85% identical to the reference sequence based on the sEQUENTIAL IDENTIFICATION NUMBER: 2, 4 or 86 having the following characteristics: the corresponding X 94 residue is threonine; the residue corresponding to X 199 is histidine and residue corresponding to X 202 is valine or leucine; and (ii) in which the residue corresponding to X 94 is alanine or threonine; the residue corresponding to X 199 is alanine, histidine or asparagine; and residue corresponding to X 202 is valine or leucine.

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Research paper thumbnail of Variants de cellobiohydrolase

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Research paper thumbnail of Biocatalyseurs et Procédés D'Hydroxylation De Composés Chimiques

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Enhanced production of recombinant Mycobacterium tuberculosis antigen 85B in Salmonella by replacement of low-usage codons

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model

Infection and Immunity, 1997

Isogeneic bacterial strains that differ only in the production of a single microbial factor have ... more Isogeneic bacterial strains that differ only in the production of a single microbial factor have been invaluable in studying the pathogenesis of bacterial infections. The targeted, intentional inactivation of a gene encoding a potential virulence determinant generally requires homologous recombination to replace the gene with an inactivated allele. To determine whether the insertion and expression of a fragment of a bacterial gene in an antisense orientation could be used as a rapid alternative to allelic inactivation for producing paired isogeneic isolates, we inverted a 600-bp fragment of the Staphylococcus aureus gene encoding alpha-toxin, hla, behind its native promoter on an Escherichia coli-S. aureus shuttle vector. A transformant of an S. aureus strain carrying the antisense hla fragment produced antisense hla RNA and made 16-fold less alpha-toxin than either its parent or an isogeneic transformant containing vector DNA without hla. Also, intraperitoneal injection of 1.5 x 10...

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Research paper thumbnail of Characterization of a Chromosomal Gene Encoding Type B β-Lactamase in Phage Group II Isolates ofStaphylococcus aureus

Antimicrobial Agents and Chemotherapy, 1998

In contrast to most Staphylococcus aureus isolates in which the gene for staphylococcal β-lactama... more In contrast to most Staphylococcus aureus isolates in which the gene for staphylococcal β-lactamase (blaZ) is plasmid borne, isolates typeable by group II bacteriophages frequently carry blaZ on the chromosome. Furthermore, the chromosomal gene encodes the type B variant of staphylococcal β-lactamase for which the nucleotide and deduced amino acid sequences have not yet been reported. To better understand β-lactamase production among phage group II staphylococci and the nature of the type B β-lactamase, we determined the type and amount of enzyme produced by 24 phage group II isolates. Of these isolates, 1 did not produce β-lactamase, 8 produced the type B enzyme, and 15 produced the type C enzyme. In all eight type B β-lactamase-producing isolates, blaZ was located on the chromosome. This was in contrast to the type C β-lactamase-producing isolates, in which blaZ was located on a 21-kb plasmid. The nucleotide sequence corresponding to the leader peptide and the N-terminal 85% of th...

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Research paper thumbnail of Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216

Journal of Bacteriology, 1996

beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lac... more beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of Staphylococcus aureus beta-lactamase, designated A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in primary structure by only a few amino acids. Using the reported sequences of the A, C, and D enzymes along with crystallographic data about the structure of the type A enzyme to identify amino acid differences located close to the active site, we hypothesized that these differences might explain the kinetic heterogeneity of the wild-type beta-lactamases. To test this hypothesis, genes encoding the type A, C, and D beta-lactamases were modified by site-directed mutagenesis, yielding mutant enzymes with single amino acid substitutions. The substitution of asparagine for serine at residue 216 of type A beta-lactamase resulted in a kinetic profi...

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Research paper thumbnail of Improved Ketoreductase Polypeptides for the Stereoselective Production of (R)-3-HYDROXYTHIOLANE

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Polynucleotides Encoding Engineered Ketoreductase Polypeptides

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Ketoreductase polypeptides and uses thereof

The present disclosure provides ketoreductase enzymes that have been engineered with wild-type ke... more The present disclosure provides ketoreductase enzymes that have been engineered with wild-type ketoreductase enzyme and improved properties compared to naturally occurring. Polynucleotides encoding the engineered ketoreductase enzyme, the host cell capable of expressing the engineered ketoreductase enzyme, and methods of synthesizing the various chiral compounds using the engineered ketoreductase enzyme also provided. In one aspect, the present disclosure is 2- [3- [3- [2- (7-chloro-2-quinolinyl) ethenyl] phenyl] -3-oxopropyl] benzoic acid methyl ester ( "substrate") ( S, E) - can be reduced to methyl 2- (3- (3- (2- (7-chloro-2-yl) vinyl) phenyl) -3-hydroxypropyl) benzoate ( "product") or to provide a ketoreductase polypeptide capable of converting.

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Research paper thumbnail of Ketoreductase Polypeptides for the Production of Azetidinone

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Research paper thumbnail of Highly Enantioselective Reduction of a Small Heterocyclic Ketone: Biocatalytic Reduction of Tetrahydrothiophene-3-one to the Corresponding (R)-Alcohol

Organic Process Research & Development, 2010

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Research paper thumbnail of Highly Enantioselective Reduction of a Small Heterocyclic Ketone: Biocatalytic Reduction of Tetrahydrothiophene-3-one to the Corresponding (R)-Alcohol

Organic Process Research & Development, 2010

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Research paper thumbnail of Development of a Practical Biocatalytic Process for ( R )-2-Methylpentanol

Organic Process Research & Development, 2010

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Research paper thumbnail of Growth of Staphylococcus aureus with Nafcillin In Vitro Induces  -Toxin Production and Increases the Lethal Activity of Sterile Broth Filtrates in a Murine Model

Journal of Infectious Diseases, 1995

The morbidity and mortality of Staphylococcus aureus infections remain high despite antibiotic th... more The morbidity and mortality of Staphylococcus aureus infections remain high despite antibiotic therapy. To investigate further the observation that penicillins increase the hemolytic activity of staphylococcal cultures, 37 strains were grown in broth with and without subinhibitory nafcillin. Nafcillin stimulated hemolytic activity in nafcillin-susceptible and -resistant isolates. Sterile broth filtrates of nafcillin-associated cultures injected intraperitoneally in mice were more rapidly lethal than filtrates of the same strain grown without nafcillin. Lethality was neutralized by anti-alpha-toxin antisera. DNA-RNA hybridization revealed a nafcillin-associated increase in alpha-toxin mRNA during the postexponential growth phase after the activation of agr. Isolates grown in slightly inhibitory nafcillin concentrations had more alpha-toxin mRNA than did nafcillin-free cultures, whereas agr RNAIII levels were comparable. This suggests that nafcillin-induced alpha-toxin production is not entirely attributable to agr. A supplemental regulatory mechanism may be involved.

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Research paper thumbnail of β‐Lactamase Production Diminishes the Prophylactic Efficacy of Ampicillin and Cefazolin in a Guinea Pig Model of Staphylococcus aureus Wound Infection

The Journal of Infectious Diseases, 1998

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Research paper thumbnail of Enhanced Production of Recombinant Mycobacterium tuberculosis Antigens in Escherichia coli by Replacement of Low-Usage Codons

Infection and Immunity, 2000

A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is t... more A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is the inability to produce large quantities of these proteins. To test the hypothesis that poor expression of some mycobacterial genes in Escherichia coli is due, in part, to the presence of low-usage E. coli codons, we used site-directed mutagenesis to convert low-usage codons to high-usage codons for the same amino acid in the Mycobacterium tuberculosis genes for antigens 85A and 85B and superoxide dismutase. Replacement of five codons in the wild-type gene for antigen 85B increased recombinant protein production in E. coli 54-fold. The recombinant antigen elicited proliferation and gamma interferon production by lymphocytes from healthy tuberculin reactors and was recognized by monoclonal antibodies to native antigen 85, indicating that the recombinant antigen contained T-cell and B-cell epitopes. Northern blotting demonstrated only a 1.7- to 2.5-fold increase in antigen 85B mRNA, sugges...

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Research paper thumbnail of Iron-cofactored Superoxide Dismutase Inhibits Host Responses toMycobacterium tuberculosis

American Journal of Respiratory and Critical Care Medicine, 2001

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