title: The role of a conserved histidine residue, His324, in Trigonopsis variabilis D-amino acid oxidase.
abstract: To investigate the functional role of an invariant histidine residue in Trigonopsis variabilis D-amino acid oxidase (DAAO), a set of mutant enzymes with replacement of the histidine residue at position 324 was constructed and their enzymatic properties were examined. Wild-type and mutant enzymes have 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 immuno-identical with that of the 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, His324/Asn/Gln/Ala/Tyr/Glu, exhibited 38-181% increase in Km and a 2-10-fold reduction in kcat/Km. Based on the crystal structure of a homologous protein, pig kidney DAAO, it is suggested that His324 might play a structural role for proper catalytic function of T. variabilis DAAO.
authors.affiliation: Department of Food Science and Nutrition, Hung Kuang Institute of Technology, Taichung, Taiwan.
authors.forename: L L
authors.initials: LL
authors.lastname: Lin
authors.forename: W C
authors.initials: WC
authors.lastname: Wang
authors.forename: S S
authors.initials: SS
authors.lastname: Ju
authors.forename: H R
authors.initials: HR
authors.lastname: Chien
authors.forename: W H
authors.initials: WH
authors.lastname: Hsu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D004926:Escherichia coli; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D003904:Mitosporic Fungi / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D003605:D-Amino-Acid Oxidase