Possible role of a histidine residue in the substrate specificity of yeast d-aspartate oxidase.

ABSTRACT

D-Aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic D-amino acids, whereas neutral and basic D-amino acids are substrates of D-amino acid oxidase (DAO). DDO of the yeast Cryptococcus humicola (ChDDO) has much higher substrate specificity to D-aspartate, but the structural features that confer this specificity have not been elucidated. A three-dimensional model of ChDDO suggested that a histidine residue (His56) in the active site might be involved in the unique substrate specificity, possibly through the interaction with the substrate side chain in the active site. His56 mutants with several different amino acid residues (H56A, H56D, H56F, H56K and H56N) exhibited no significant activity toward acidic D-amino acids, but H56A and H56N mutants gained the ability to utilize neutral D-amino acids as substrates, such as D-methionine, D-phenylalanine and D-glutamine, showing the conversion of ChDDO to DAO by these mutations. This conversion was also demonstrated by the sensitivity of these mutants to competitive inhibitors of DAO. These results and kinetic properties of the mutants show that His56 is involved in the substrate specificity of ChDDO and possibly plays a role in the higher substrate specificity toward D-aspartate.