Catalytic and structural characteristics of carp hepatopancreas D-amino acid oxidase expressed in Escherichia coli.
D-amino acid oxidase of carp (Cyprinus carpio) hepatopancreas was overexpressed in Escherichia coli cells and purified to homogeneity for the first time in animal tissues other than pig kidney. The purified preparation had a specific activity of 293 units mg(-1) protein toward D-alanine as a substrate. It showed the highest activity toward D-alanine with a low Km of 0.23 mM and a high kcat of 190 s(-1) compared to 10 s(-1) of the pig kidney enzyme. Nonpolar and polar uncharged D-amino acids were preferable substrates to negatively or positively charged amino acids. The enzyme exhibited better thermal and pH stabilities than several yeast counterparts or the pig kidney enzyme. Secondary structure topology consisted of 11 alpha-helices and 17 beta-strands that differed slightly from pig kidney and Rhodotorula gracilis enzymes. A three-dimensional model of the carp enzyme constructed from a deduced amino acid sequence resembled that of pig kidney D-amino acid oxidase but with a shorter active site loop and a longer C-terminal loop. Judging from these characteristics, carp D-amino acid oxidase is close to the pig kidney enzyme structurally, but analogous to the R. gracilis enzyme enzymatically in turnover rate and pH and temperature stabilities.
Sarower
Mohammed Golam
MG
Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.
Okada
Shigeru
S
Abe
Hiroki
H
D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002347:Carps / Q000378:metabolism*; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D043143:Hepatopancreas / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013696:Temperature
D004791:Enzyme Inhibitors; D003605:D-Amino-Acid Oxidase; D000409:Alanine