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R Tata

Publications and source records attributed to R Tata.

16 recordsLinked to original sources
PubMed2003

Burkholderia genome analysis reveals new enzymes belonging to the nitrilase superfamily. The amidase of Burkholderia cepacia (hospital isolate).

Burkholderia cepacia (formerly Pseudomonas cepacia) grows in media containing acetamide or propionamide as C and N sources. Chromosomal DNA from a hospital isolate of B. cepacia served as a template in PCRs using primers designed for the amplification of the P. aeruginosa amiE gene that encodes an aliphatic amidase. Partial sequencing of the PCR products gave a translated sequence 100% identical with the amino acid sequence of P. aeruginosa amidase. A search of Burkholderia genomes detected a putative amidase in B. cepacia J2315 with high identity to the P. aeruginosa amidase and predicted that other Burkholderia species also possessed CN_hydrolases that use the same catalytic triad (Glu-Lys-Cys) as amidase. Superimposition of theoretical three-dimensional models suggested that differences in the amino acid sequences between amidases from B. cepacia (hospital isolate) and B. cepacia J2315 do not affect their three-dimensional structure.

Amidohydrolases↗
PubMed2001

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability.

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55 degrees C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instability due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M(r) value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.

Amidohydrolases↗
PubMed2000

Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme.

A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70-80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M(r) of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.

Amidohydrolases↗
PubMed1999

Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme.

Wild-type and site-specific mutants C166S and C166A (Cys-166-->Ser and Cys-166-->Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a=b=c=84 A; alpha=beta=gamma=75 degrees) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

Amidohydrolases↗
PubMed1995

Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys166 is predicted to be the active site nucleophile of the catalytic mechanism.

A database search indicated homology between some members of the nitrilase/cyanide hydratase family, Pseudomonas aeruginosa and Rhodococcus erythropolis amidases and several other proteins, some of unknown function. BLOCK and PROFILE searches confirmed these relationships and showed that four regions of the P. aeruginosa amidase had significant homology with corresponding regions of nitrilases. A phylogenetic tree placed the P. aeruginosa and R. erythropolis amidases in a group with nitrilases but separated other amidases into three groups. The active site cysteine in nitrilases is conserved in the P. aeruginosa amidase indicating that Cys166 is the active site nucleophile.

Amidohydrolases↗
PubMed1994

Arg-188 and Trp-144 are implicated in the binding of urea and acetamide to the active site of the amidase from Pseudomonas aeruginosa.

Urea is a time-dependent active-site-directed inhibitor of Pseudomonas aeruginosa amidase. We found that 20 mM hydroxylamine caused bound urea to be released from the inactive urea:amidase complex with the restoration of enzyme activity. Bound urea restricts the titrability of the enzyme's -SH groups to 6 per hexameric molecule and protects it against thermal denaturation suggesting that urea binding provokes a conformational change in the enzyme. Mutations in the P. aeruginosa amidase gene that reduce the binding affinity of the enzyme for both urea and the substrate acetamide have been identified by direct sequencing of PCR-amplified mutant genes and confirmed by sequencing cloned PCR-amplified genes. The mutations were in two regions of the enzyme substituting either Arg-188 (or Gln-190, in one case) or Trp-144; one amidase that bound neither urea nor acetamide was doubly mutant with an amino-acid change at both sites.

Acetamides↗
PubMed1989

Dielectric behavior of aqueous solutions of plasmid DNA at microwave frequencies.

The relative permittivity and dielectric loss of aqueous solutions of plasmid (pUC8.c1 and pUC8.c2) DNA have been measured at 20 degrees C over the frequency range 100 MHz-10 GHz. The solutions had a concentration of 0.1% DNA, and were studied both in the relaxed and the supercoiled form. The dielectric measurements were made using a variety of techniques including frequency domain and time domain methods of operation. No evidence of any resonance absorption, nor of any other kind of enhanced absorption, was observed.

DNA, Superhelical↗
PubMed1988

Mutations affecting the synthesis of NADP-dependent glutamate dehydrogenase in Pseudomonas aeruginosa.

NADP-dependent glutamate dehydrogenase (NADP-GDH) was purified to homogeneity from Pseudomonas aeruginosa strain 8602 (PAC 1). The Mr determined by Sephadex gel filtration was 280,000; the subunit Mr determined by SDS-PAGE was 45,000. Mutant strains lacking NADP-GDH and glutamate synthase (Gdh-Glt-) required glutamate for growth. Transductants that lacked only NADP-GDH were indistinguishable from the wild-type strain in growth properties. It was concluded that NADP-GDH is not essential for growth of the wild-type organism and that glutamate formation via NAD-dependent glutamate dehydrogenase does not occur to a significant extent. A mutant strain, 39, producing high NADP-GDH activity, synthesized normal NADP-GDH and had the same intracellular glutamate concentrations as its parent. The mutation responsible for the synthesis of high levels of NADP-GDH was shown, by transduction, to be closely linked to the NADP-GDH structural gene (gdhA).

Genetic Linkage↗
PubMed1987

Glycollate inhibition of growth of Pseudomonas aeruginosa on lactate medium.

Glycollate inhibited growth of Pseudomonas aeruginosa in media containing either pyruvate or lactate as carbon sources. Glycollamide, but not glyoxylate, showed similar effects. Spontaneous mutants (L/G strains) were isolated that were able to grow on lactate medium in the presence of glycollate: their growth in pyruvate medium was still inhibited by glycollate. Synthesis of membrane-bound NAD+-independent D(-)- and L(+)-lactate dehydrogenase (iLDHs) was inducible by D- or L-lactate in the parent strain but was constitutive in the L/G strains. Glycollate inhibited induction of the synthesis of iLDHs in the parent strain growing in succinate medium but had no effect under the same conditions on strain L/G1. Glycollate was a competitive inhibitor of L(+)-iLDH (Ki = 11 mM). No differences were found in the kinetic properties of L(+)-iLDH in cell-free extracts from strain L/G1 and the parent organism. Glycollate appears to inhibit growth on lactate medium predominantly through prevention of lactate induction of iLDH synthesis.

Enzyme Induction↗
PubMed1987

Isolation of amidase-negative mutants of Pseudomonas aeruginosa using glycollamide as a selective agent.

A positive selection is described for isolating amidase-negative mutants from Pseudomonas aeruginosa strains. The method is based on the conversion, via amidase activity, of glycollamide to glycollate which is growth inhibitory. Three types of mutant were isolated on lactate medium containing glycollamide: (i) mutants in which amidase activity was reduced or absent; (ii) double mutants that were amidase-negative and resistant to glycollate inhibition of growth; and (iii) glycollate-resistant mutants. By raising glycollamide concentrations in the selection medium, amidase-negative mutants were obtained from strains producing altered amidases with low specific acetamidase and glycollamidase activities. Glycollamide has wider applicability than fluoroacetamide as a selective agent for obtaining amidase-negative mutants.

Amidohydrolases↗
PubMed1981

Growth of Pseudomonas aeruginosa mutants lacking glutamate synthase activity.

Mutant strains SU1, SU4, and US1 lacking glutamate synthase (GOGAT) activity were isolated from strains of P. aeruginosa for which histidine is a growth rate-limiting source of nitrogen. Strains SU1 and SU4 were unable to grow when a low concentration of ammonia and a variety of compounds, including histidine, were supplied as sole sources of nitrogen. A revertant of strain SU1, strain 39, produced no GOGAT but high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase and had restored ability to grow on a limited number of nitrogen sources. Strain US1 grew at the same rate in histidine medium as did its parent; it was derepressed for glutamine synthase synthesis, and histidase was less sensitive to repression by ammonia than in the parent strain. We conclude that GOGAT is not essential for growth on histidine but high levels of glutamine synthase are required nd high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase can sustain growth at low concentrations of ammonia in the absence of GOGAT.

Ammonia↗
PubMed1978

Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor.

Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI3. AI3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2. Compared with AI3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.

Amidohydrolases↗
PubMed1977

Pseudomonas aeruginosa mutants resistant to urea inhibition of growth on acetanilide.

Pseudomonas aeruginosa AI 3 was able to grow in medium containing acetanilide (N-phenylacetamide) as a carbon source when NH4+ was the nitrogen source but not when urea was the nitrogen source. AIU mutants isolated from strain AI 3 grew on either medium. Urease levels in bacteria grown in the presence of urea were 10-fold lower when NH4+ or acetanilide was also in the medium, but there were no apparent differences in urease or its synthesis between strain AI 3 and mutant AIU 1N. The first metabolic step in the acetanilide utlization is catalyzed by an amidase. Amidases in several AIU strains showed altered physiochemical properties. Urea inhibited amidase in a time-dependent reaction, but the rates of the inhibitory reaction with amidases from the AIU mutants were slower than with AI 3 amidase. The purified amidase from AIU 1N showed a marked difference in its pH/activity profile from that obtained with purified AI 3 amidase. These observations indicate that the ability of strain AIU 1N and the other mutants to grow on acetanilide/urea medium is associated with a mutation in the amidase structural gene; this was confirmed for strain AIU 1N by transduction.

Acetanilides↗
PubMedDate not supplied

Microwave absorption in aqueous solutions of DNA.

In a recent Nature leading article the possibility of the occurrence of resonance absorption at microwave frequencies in DNA was discussed in terms of the latest available theoretical and experimental evidence, and its implications were assessed. Because of the far-reaching nature of these implications it is important that the existence or otherwise of such absorption be firmly established. Here we report on a concerted effort in two independent laboratories which has involved the measurement of the dielectric properties of aqueous solutions of circular DNA molecules in the frequency range 1-10 GHz. No resonance absorption or any form of enhanced absorption was demonstrated.

DNA, Bacterial↗