Engineering the substrate specificity of porcine kidney D-amino acid oxidase by mutagenesis of the "active-site lid".
Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at I215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which I215-N225 is substituted by R216-G220 of DDO, showed D-aspartate-oxidizing activity that wild-type DAO does not exhibit, together with a considerable decrease in activity toward D-alanine. These findings indicate that I215-N225 of DAO contributes profoundly to its substrate specificity. Based on these results and the crystal structure of DAO, we systematically mutated the E220-Y224 region within the short stretch in question and obtained five mutants (220D224G, 221D224G, 222D224G, 223D224G, and 224D), in each of which an aspartate residue is mutated to E220-Y224. All of the mutants exhibited decreased apparent K(m) values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, k(cat app)/K(m app), for D-arginine increased by one order of magnitude for the 221D224G or 222D224G mutant, whereas that for D-alanine or D-serine decreased to marginal or nil.
Setoyama
Chiaki
C
Departments of Molecular Enzymology and Molecular Physiology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556.
Nishina
Yasuzo
Y
Mizutani
Hisashi
H
Miyahara
Ikuko
I
Hirotsu
Ken
K
Kamiya
Nobuo
N
Shiga
Kiyoshi
K
Miura
Retsu
R
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D050804:D-Aspartate Oxidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D013552:Swine
D011994:Recombinant Proteins; D050804:D-Aspartate Oxidase; D003605:D-Amino-Acid Oxidase