Renée Tata

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.

Mechanism and consequence of the autoactivation of p38α mitogen-activated protein kinase promoted by TAB1

p38α mitogen-activated protein kinase (p38α) is activated by a variety of mechanisms, including autophosphorylation initiated by TGFβ-activated kinase 1 binding protein 1 (TAB1) during myocardial ischemia and other stresses. Chemical-genetic approaches and coexpression in mammalian, bacterial and cell-free systems revealed that mouse p38α autophosphorylation occurs in cis by direct interaction with TAB1(371–416). In isolated rat cardiac myocytes and perfused mouse hearts, TAT-TAB1(371–416) rapidly activates p38 and profoundly perturbs function. Crystal structures and characterization in solution revealed a bipartite docking site for TAB1 in the p38α C-terminal kinase lobe. TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif, allowing autophosphorylation in cis. Interference with p38α recognition by TAB1 abolishes its cardiac toxicity. Such intervention could potentially circumvent the drawbacks of clinical pharmacological inhibitors of p38 catalytic activity.

Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity.

The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys(166) is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase-fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.

Heterodimerization of the human RNase P/MRP subunits Rpp20 and Rpp25 is a prerequisite for interaction with the P3 arm of RNase MRP RNA

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20–Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition.

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°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its Mr value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.

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 enzymes -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.

Isolation of Amidase-negative Mutants of Pseudomonas aeruginosa by a Positive Selection Method Using an Acetamide Analogue

Pseudonionas aeruginosa is resistant to many amino acid and purine and pyrimidine analogueswhich causegrowth inhibition of Escherichiacoliand this has precluded the use of such analogues for the isolation of mutants either resistant to feedback repression or derepressed for the synthesis of biosynthetic enzymes (Holloway, 1969). Calhoun & Jensen (1972) were able to increase the sensitivity of P. aeruginosa to certain metabolic analogues by changing the carbon source in the growth medium. p-Amino-phenylalanine and /j-a-thienylalanine did not inhibit the growth of P. aeruginosa on minimal agar plates with glucose as the carbon source but with fructose there was pronounced growth inhibition and resistant colonies were isolated from the inhibition zones after a few days of incubation. Calhoun & Jensen (I 972) suggest that the metabolic pool of cells growing on fructose is relatively low in the precursors for the synthesis of aromatic amino acids so that analogues of these acids are able to compete successfully for certain enzymes and exert a growth inhibitory effect. These results suggest that if growth media can be devised which decrease the metabolic pools of precursors of other amino acids it might be possible to increase the sensitivity of P. aerriginosa to metabolic analogues and thereby isolate regulatory mutants. Leisinger, Haas & Hegarty (1972) found that P. aeruginosa was more sensitive to growth inhibition by indospicine (an analogue of arginine) when the cultures were grown on an ornithine medium, than in a glucose + arginine medium. With 2 mM-indospicine the doubling time in ornithine medium was increased eightfold from the control culture while in the glucose + arginine medium the increase was only twofold. With 2 mwcanavanine the mean generation time was increased twofold in both media. For catabolic enzymes it could be predicted that if a substrate analogue could give rise to a toxic product it should be possible to increase the sensitivity of the culture to the analogue by arranging growth conditions so that the bacteria contained a high level of the enzyme concerned. Hynes & Pateman (1970) found that mutants of Aspergillus rzidulatis which produced acetamidase constitutively were more sensitive to growth inhibition by fluoracetamide than was the wild-type strain. They were able to isolate acetamide-negative mutants from a semiconstitutive mutant plated on an acetate medium containing 2 mg fluorace, tamide/ml after treatment of a conidial suspension with N-methyl-N’-nitro-N-nitrosoguanidine. We have adapted this method to enable us to isolate amidase-negative mutants of Pseudomonas aeruginosa by using growth media which allow the maximum rate of amidase synthesis by the parental strains. Pseudomonas aeruginosa PAC I produces an aliphatic amidase which is induced by acetamide and propionaniide and allows these two amides to be used as the carbon and nitrogen sources for growth. Constitutive mutants have been isolated on S/F plates containing sodium succinate (I (;;) and formamide (0.1 (:/o). Formamide is a poor substrate and very

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 Mr 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.

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

SummaryHydroxyurea 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 AI 3. AI 3 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 AI 3 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.

Crystal structure of a putative phosphinothricin acetyltransferase (PA4866) from Pseudomonas aeruginosa PAC1.

Introduction. In the genome of P. aeruginosa PAO 1 (http://www.pseudomonas.com/) the gene PA4866 encodes a 172 amino acid protein (pita) belonging to the GCN5 family of acetyl transferases (PF00583). Blast searches reveal similar enzymes in a wide range of Gram-negative and Gram-positive organisms. These enzymes are listed either as hypothetical proteins or as putative phosphinothricin acetyl transferases (PAT). PAT is expressed in Streptomyces hygroscopicus and S. viridochromogenes, and catalyzes the acetylation of phosphinothricin, a glutamate analog widely used as a herbicide, which exerts its phytotoxic effect on plants by inhibiting glutamine synthetase. Pita displays 45 and 42% sequence similarity (37 and 35% sequence identity) with the two PAT enzymes, respectively. We now report the crystal structure of this enzyme, as a basis for investigating its function.