Microbial oxidases of acidic d-amino acids

Two microbial oxidases of acidic d-amino acids have been purified to homogeneity. One is a d-aspartate oxidase of the yeast Cryptococcus humicolus UJ1 that was induced markedly with d-aspartate and is far more active toward d-aspartate than d-glutamate. The other is a d-glutamate oxidase of Candida boidinii 2201 that preferred d-glutamate to d-aspartate as a substrate in terms of kcat/Km, but was not induced very effectively by d-glutamate. The most potent competitive inhibitor of the C. humicolus d-aspartate oxidase was malonate, and that of the C. boidinii d-glutamate oxidase was d-malate. The former enzyme was a homotetramer of 160 kDa consisting of subunits of 40 kDa, each of which contained 1 mol of FAD, while the latter was a monomer of 45 kDa. The N-terminal sequences of both enzymes were similar to those of other FAD enzymes and contained a consensus sequence common to most enzymes binding ADP-containing nucleotides. Peroxisomal localization of the C. humicolus d-aspartate oxidase was shown by subcellular fractionation and morphological analysis via electron microscopy of C. humicolus cells, where induction of the enzyme was accompanied by induction of catalase and development of peroxisomes. The apo-form of C. humicolus d-aspartate oxidase, prepared by removal of FAD was a monomeric protein of 40 kDa, and its binding with FAD proceeded in two stages. The Kd for the apoprotein-FAD complex was very low (8.2×10−12 M) consistent with the observed tight binding. The C. humicolus d-aspartate oxidase was essentially similar to other flavoprotein oxidases of acidic and neutral d-amino acids with respect to its spectral properties and sensitivity to specific modifying reagents for arginyl and histidyl residues.