Kazuhiro MARUYAMA

Mutational analysis of substrate binding site of the yeast D-aspartate oxidase

Ryo-hei YAMADA, Yoshiki KERA, Shouji TAKAHASHI

D-Aspartate oxidase (DDO) specifically catalyzes the oxidative deamination of acidic D-amino acids. To determine a key residue for the substrate specificity of DDO, we predicted and mutated putative substrate selection residues in DDO from the yeast Cryptococcus humicola (ChDDO).
We predicted the key residues, Arg230 or Arg243, that are likely to interact with the carboxyl group of the side chain of the substrates. To determine which residues are involved in the interaction, we individually mutated the Args to Ala. The wild-type ( ChDDOHis ) and mutant ChDDOs ( R230A ChDDOHis , R243A ChDDOHis ) were expressed as His-tag fusion proteins in E. coli and were purified by metal affinity chromatography followed by gel filtration chromatography.
The specific activities of these proteins for D-aspartate were 101 U/mg for ChDDOHis, 13.8 U/mg for R230A ChDDOHis and 0.05 U/mg for R243A ChDDOHis. In comparison with ChDDOHis, these mutations caused 5.4�`5.8 fold increase in Km for D-aspartate.
The purified ChDDOHis and mutant ChDDOHiss exhibited a typical absorption spectrum of flavoprotein. Addition of an excess of D-aspartate showed a rapid reduction of flavin ChDDOHis and R230A ChDDOHis but not R243A ChDDOHis. Addition of malonate produced certain spectral changes including red shift in ChDDOHis and R230A ChDDOHis but not in R243A ChDDOHis. These results suggest that Arg243 plays a important role in the substrate selectivity of ChDDO.