Toshiyuki TAKAHASHI

Characterization of the yeast Cryptococcus humicola UJ1 D-aspartate oxidase expressed in E. coli

Ryo-hei YAMADA, Yoshio KERA, Shouji TAKAHASHI


D-Aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic D-amino acids, while D-amino acid oxidase (DAO) catalyzes the same reaction of neutral and basic D-amino acids. In contrast to DAO, the crystal structure of DDO has not yet been determined, and the certain active site residues are not entirely clear. To study the structure-function relationship of DDO, we constructed an expression system for DDO from the yeast Cryptococcus humicola UJ1 (ChDDO) in E. coli and purified and characterized the recombinant enzyme.

The cDNA encoding ChDDO was inserted into pET11b and expressed in E. coli Rosetta (DE3) having the rare tRNA genes. The crude extract from E. coli transformant cells showed DDO activity, in which the specific activity was higher than that from the yeast. The purified recombinant enzyme exhibited the same enzymatic properties as the native enzyme as follows.The recombinant enzyme is a homotetramer by its behavior on gel filtration. Optimum pH and temperature are pH 7.5 and 37��C, respectively. The recombinant enzyme loses its activity when it keeps at 50��C for 10 min. D-Glutamate is a very poor substrate for the enzyme. N-Methyl-D-aspartate is better than D-glutamate as substrate but markedly poorer than D-aspartate. Malonate is the most effective competitive inhibitor of the compounds tested.