Engineering the substrate specificity of porcine kidney D-Amino acid oxidase by mutagenesis of the 'Active-Site Lid'

摘要

Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at 1215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which 1215-N225 is substituted by R216-G220 of DDO, showed D-aspartate-oxidizing activity that wild-type DAO does not exhibit, together with a considerable decrease in activity toward D-alanine. These findings indicate that 1215-N225 of DAO contributes profoundly to its substrate specificity. Based on these results and the crystal structure of DAO, we systematically mutated the E220-Y224 region within the short stretch in question and obtained five mutants (220D224G, 221D224G, 222D224G, 223D224G, and 224D), in each of which an aspartate residue is mutated to E220-Y224. All of the mutants exhibited decreased apparent Km values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, h(cat) (app)/K-m (app) for D-arginine increased by one order of magnitude for the 221D224G or 222D224G mutant, whereas that for D-alanine or D-serine decreased to marginal or nil.