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.
Novo
Carlos
C
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
Farnaud
Sebastien
S
Tata
Renée
R
Clemente
Alda
A
Brown
Paul R
PR
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
D000581:Amidohydrolases; C019665:amidase