Alteration of substrate specificity of fructosyl-amino acid oxidase from Fusarium oxysporum.
Fructosyl-amino acid oxidase (FOD-F) from Fusarium oxysporum f. sp. raphani (NBRC 9972) is the enzyme catalyzing the oxidative deglycation of fructosyl-amino acids such as N(epsilon)-fructosyl N(alpha)-benzyloxycarbonyl-lysine (FZK) and fructosyl valine (FV), which are model compounds of the glycated proteins in blood. Wild-type FOD-F has high activities toward both substrates. We obtained a mutant FOD-F, which reacts with FZK but not with FV by random mutagenesis. One amino-acid substitution (K373R) occurred in the mutant FOD-F. In addition to K373R, K373W, K373M, K373T, and K373V, which were selected for optimization of the substitution at position K373, were purified and characterized. Kinetic analysis showed that the catalytic turnover for FV greatly decreased, whereas that for FZK did not. In consequence, the specificities toward FZK were increased in the mutant FOD-Fs. The relation between the substrate specificity of the mutant FOD-Fs and the position of the carboxyl group of the substrates was demonstrated using a series of the substrates having the carboxyl group at the different position. The mutant FOD-Fs are attractive candidates for developing an enzymatic measurement method for glycated proteins such as glycated albumin in serum. This study will be helpful to establish an easier and rapid clinical assay system of glycated albumin.
Fujiwara
Maki
M
Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
Sumitani
Jun-ichi
J
Koga
Shinji
S
Yoshioka
Issei
I
Kouzuma
Takuji
T
Imamura
Shigeyuki
S
Kawaguchi
Takashi
T
Arai
Motoo
M
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D005670:Fusarium / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020125:Mutation, Missense; D013379:Substrate Specificity / Q000235:genetics*; D014633:Valine / Q000031:analogs & derivatives / Q000378:metabolism
C045420:fructosylvaline; C033186:fructosyl-lysine; D000594:Amino Acid Oxidoreductases; C103012:amadoriase; D014633:Valine; D008239:Lysine