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Journal of Biochemistry 2006 139(5):873-879; doi:10.1093/jb/mvj094
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© 2006 The Japanese Biochemical Society.

Regular Paper

Engineering the Substrate Specificity of Porcine Kidney D-Amino Acid Oxidase by Mutagenesis of the "Active-Site Lid"

Chiaki Setoyama1, Yasuzo Nishina2, Hisashi Mizutani3, Ikuko Miyahara3, Ken Hirotsu3, Nobuo Kamiya3, Kiyoshi Shiga2 and Retsu Miura1,*

Departments of 1 Molecular Enzymology and 2 Molecular Physiology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556; and 3 Department of Chemistry, Graduate School of Science, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka 558;8585

* To whom correspondence should be addressed. Fax: +81-96-373-5066, E-mail: miura{at}gpo.kumamoto-u.ac.jp

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 Km values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, kcat app/Km 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.


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Functional and Structural Characterization of D-Aspartate Oxidase from Porcine Kidney: Non-Michaelis Kinetics due to Substrate Activation
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