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

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.

Structural Insights into the Catalytic Active Site and Activity of Human Nit2/ω-Amidase

“…All enzymes of the nitrilase family contain a highly conserved catalytic Glu-Lys-Cys triad in the active site (24,27). Novo et al (28) reported that mutation of any residue of this triad will result in loss of aliphatic amidase activity in Pseudomonas aeruginosa. The crystal structures of Caenorhabditis elegans NitFhit (nitrilase-fragile histidine fusion protein) (25), Saccharomyces cerevisiae Nit3 (29), and mouse Nit2 (mNit2) (26) have been determined.…”

Structure of Amidase from Pseudomonas aeruginosa Showing a Trapped Acyl Transfer Reaction Intermediate State

“…The oligomer stability must therefore derive from other types of stabilization factors. Other research workers (20,26) have reported the structural role of Glu 59 in the maintenance of the quaternary structure of the enzyme because the substitution Glu 59 3 Val resulted in the dissociation of hexamer into dimers with loss of amidase activity. The hexameric arrangement described above together with the tight packing through the long C-terminal arm embracement, unique among known structural homologues, may constitute the relevant factors promoting the observed remarkable thermostability of amidase.…”

“…Although the catalytic mechanism is still not fully understood (24 -26), this enzyme has attracted great attention because of the variations in its substrate and inhibitor specificities that can be generated through single point mutations. Cys 166 has been found to act as the nucleophile in the covalent catalysis of P. aeruginosa amidase, within a catalytic triad, Glu-Lys-Cys, that has been consistently identified in all members of the nitrilase superfamily (13,26).…”

Crystallization, diffraction data collection and preliminary crystallographic analysis of hexagonal crystals ofPseudomonas aeruginosaamidase

“…Although the detailed catalytic mechanism is still not totally understood (Findlater & Orsi, 1973;Woods et al, 1979;Novo et al, 2002), this enzyme has attracted great attention because of the variations in its substrate and inhibitor specificities that can be generated by single-point mutations (Tata et al, 1994;Karmali et al, 2000Karmali et al, , 2001Gregoriou & Brown, 1979, 1980. Microbial amidases with altered substrate specificities can be used in several industrial applications such as the detoxification of industrial effluents containing toxic amides and the production of hydroxamic acids and other organic acids (Fournand et al, 1998).…”