Syunsuke MATSUMOTO

Mutational analysis of a possible key determinant of substrate specificity of yeast D-aspartate oxidase

Ryo-hei YAMADA, Yoshiki KERA, Shouji TAKAHASHI

D-Aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic D-amino acids. Although much information is available on its biochemical properties, little is known about its structure-function relationship. We previously obtained the results suggesting that, in DDO of the yeast Cryptococcus humicola (ChDDO), Arg243 is a key residue in its strict substrate specificity for acidic D-amino acids. In this study, to clearly define the role of Arg243, we mutated the Arg residue to various amino acid residues and characterized the mutants. Arg243 in ChDDO was mutated to Asp, Glu, Met, or Lys by site-directed mutagenesis. The resulting mutants and the wild-type enzyme were expressed as His-tagged fusion proteins in Escherichia coli and were purified to homogeneity by column chromatographies. All the purified mutants showed a typical flavin adsorption spectrum as well as the wild-type enzyme but had lower activities toward D-aspartate than the wild-type, except for R243D, which showed an activity approximately twice that of the wild-type. Among them, R243M and R243E gained activity toward D-alanine and exhibited higher activity toward D-glutamate than the wild-type. In addition, in contrast to the wild-type, R243E also exhibited a slight activity toward D-arginine. These results suggested that Arg243 plays an important role in binding and selection of substrate in ChDDO.