Ll. Lin et al., The role of a conserved histidine residue, His324, in Trigonopsis variabilis D-amino acid oxidase, FEMS MICROB, 176(2), 1999, pp. 443-448
To investigate the functional role of an invariant histidine residue in Tri
gonopsis variabilis D-amino acid oxidase (DAAO), a set of mutant enzymes wi
th replacement of the histidine residue at position 324 was constructed and
their enzymatic properties were examined, Wild-type and mutant enzymes hav
e been purified to homogeneity using the His-bound column and the molecular
masses were determined to be 39.2 kDa. Western blot analysis revealed that
the in vivo synthesized mutant enzymes are immune-identical with that of t
he wild-type DAAO. The His324Asn and His324Gln mutants displayed comparable
enzymatic activity to that of the wild-type enzyme, while the other mutant
DAAOs showed markedly decreased or no detectable activity. The mutants, Hi
s324/Asn/Gln/Ala/Tyr/Glu, exhibited 38-181% increase in K-m and a 2-10-fold
reduction in k(cat)/K-m. Based on the crystal structure of a homologous pr
otein, pig kidney DAAO, it is suggested that His324 might play a structural
role for proper catalytic function of T. variabilis DAAO. (C) 1999 Federat
ion of European Microbiological Societies. Published by Elsevier Science B.
V. All rights reserved.