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Journal List > Biochem J > v.365(Pt 3); Aug 1, 2002

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Biochem J. 2002 August 1; 365(Pt 3): 731–738.
doi: 10.1042/BJ20011714.
PMCID: PMC1222709
Copyright notice
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.
Carlos Novo, Sebastien Farnaud, Renée Tata, Alda Clemente, and Paul R Brown
Unidade de Tecnologica de Proteínas e Anticorpos Monoclonais, Departmento de Biotecnologia, Instituto Nacional de Engenharia e Tecnologia Industrial, Edifício F, Estrada do Paço do Lumiar 1649-038, Lisbon, Portugal. carlos.novo@ineti.pt
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Abstract
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.
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Selected References
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