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, Paul R. Brown

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys166 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-GluLys 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.

Original languageEnglish
Pages (from-to)731-738
Number of pages8
JournalBiochemical Journal
Volume365
Issue number3
DOIs
Publication statusPublished - 1 Aug 2002
Externally publishedYes

Fingerprint

amidase
nitrilase
Mutagenesis
Substrate Specificity
Site-Directed Mutagenesis
Pseudomonas aeruginosa
Mutation
Substrates
Amidohydrolases
Nucleophiles
Histidine
Proteins
Fusion reactions
Crystal structure
Databases
Amino Acids

Keywords

  • Comparative modelling
  • NitFhit
  • Nitrilase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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. / Novo, Carlos; Farnaud, Sebastien; Tata, Renée; Clemente, Alda; Brown, Paul R.

In: Biochemical Journal, Vol. 365, No. 3, 01.08.2002, p. 731-738.

Research output: Contribution to journalArticle

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