Engineering the substrate specificity of porcine kidney D-amino acid oxidase by mutagenesis of the "active-site lid"

J Biochem. 2006 May;139(5):873-9. doi: 10.1093/jb/mvj094.

Abstract

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 I215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which I215-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 I215-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 K(m) values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, k(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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / genetics
  • Brain / metabolism
  • Cloning, Molecular
  • D-Amino-Acid Oxidase / genetics
  • D-Amino-Acid Oxidase / isolation & purification
  • D-Amino-Acid Oxidase / metabolism*
  • D-Aspartate Oxidase / genetics
  • D-Aspartate Oxidase / isolation & purification
  • D-Aspartate Oxidase / metabolism
  • Humans
  • Kidney / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Engineering
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Substrate Specificity
  • Swine

Substances

  • Recombinant Proteins
  • D-Aspartate Oxidase
  • D-Amino-Acid Oxidase