title: Catalytic mechanism of fungal homoserine transacetylase.
abstract: Homoserine transacetylase is a required catalyst in the biochemical pathway that metabolizes Asp to Met in fungi. The enzyme from the yeast Schizosaccharomyces pombe activates the hydroxyl group of L-homoserine by acetylation from acetyl coenzyme A. This enzyme is unique to fungi and some bacteria and presents an important new target for drug discovery. Steady-state kinetic parameters provide evidence that this enzyme follows a ping-pong mechanism. Proton inventory was consistent with a single-proton transfer, and pH studies suggested the participation of at least one residue with a pKa value of 6.4-6.6, possibly a His or Asp/Glu in catalysis. Protein sequence alignments indicate that this enzyme belongs to the alpha/beta-hydrolase fold superfamily of enzymes, indicating the involvement of an active-site nucleophile and possibly a canonical catalytic triad. We constructed site-specific mutants and identified Ser163, Asp403, and His432 as the likely active-site residues of a catalytic triad based on steady-state kinetics and genetic complementation of a yeast null mutant. Moreover, unlike the wild-type enzyme, inactive site mutants were not capable of producing an acetyl-enzyme intermediate. Homoserine transacetylase therefore catalyzes the acetylation of L-homoserine via a covalent acyl-enzyme intermediate through an active-site Ser. These results form the basis of future exploitation of this enzyme as an antimicrobial target.
authors.affiliation: Antimicrobial Research Centre, Department of Biochemistry and Biomedical Sciences, McMaster University, Ontario, Canada, L8N 3Z5.
authors.forename: Ishac
authors.initials: I
authors.lastname: Nazi
authors.forename: Gerard D
authors.initials: GD
authors.lastname: Wright
mesh_terms: D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D012568:Schizosaccharomyces / Q000201:enzymology*
chemical_list: D003903:Deuterium; D000123:Acetyltransferases; C021369:homoserine O-acetyltransferase