Microbial oxidases of acidic D-amino acids

Citation
R. Yamada et al., Microbial oxidases of acidic D-amino acids, J MOL CAT B, 12(1-6), 2001, pp. 93-104
Citations number
37
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Physical Chemistry/Chemical Physics
Journal title
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
ISSN journal
1381-1177 → ACNP
Volume
12
Issue
1-6
Year of publication
2001
Pages
93 - 104
Database
ISI
SICI code
1381-1177(20010228)12:1-6<93:MOOADA>2.0.ZU;2-P
Abstract
Two microbial oxidases of acidic D-amino acids have been purified to homoge neity. 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 t erms of k(cat)/K-m, but was not induced very effectively by D-glutamate. Th e 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 I mol of FAD, while the latter was a monom er of 45 kDa. The N-terminal sequences of both enzymes were similar to thos e of other FAD enzymes and contained a consensus sequence common to most en zymes 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 devel opment of peroxisomes. The ape-form of C. humicolus D-aspartate oxidase, pr epared by removal of FAD was a monomeric protein of 40 kDa, and its binding with FAD proceeded in two stages. The K-d for the apoprotein-FAD complex w as very low (8.2 x 10(-12) M) consistent with the observed tight binding. T he C. humicolus D-aspartate oxidase was essentially similar to other flavop rotein oxidases of acidic and neutral D-amino acids with respect to its spe ctral properties and sensitivity to specific modifying reagents for arginyl and histidyl residues. (C) 2001 Elsevier Science B.V. All rights reserved.