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PEDS Advance Access published online on January 31, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzm047
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis

Seiji Miura1, Stefano Ferri1, Wakako Tsugawa1, Seungsu Kim1 and Koji Sode1,2

1Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi 185-8688, Japan

2 To whom correspondence should be addressed. E-mail: sode{at}cc.tuat.ac.jp

Docking models of fructosyl amine oxidase (FAOD) from the marine yeast Pichia N1-1 (N1-1 FAOD) with the substrates fructosyl valine (f-Val) and fructosyl-{varepsilon}N-lysine (f-{varepsilon}Lys) were produced using three-dimensional protein model as reported previously (Miura et al., 2006, Biotechnol. Lett., 28, 1895-1900). The residues involved in recognition of substrates were proposed, particularly Asn354, which interacts closely with f-{varepsilon}Lys, but not with f-Val. Substitution of Asn354 to histidine and lysine simultaneously resulted in an increase in activity of f-val and a decrease in activity of f-{varepsilon}Lys and thus, increasing the specificity for f-Val from 13- to 19-fold. In addition to creating two mutant FAODs with great potential for the measurement of glycated hemoglobin, we have provided the first structural model of substrate binding with eukaryotic FAOD, which is expected to contribute to further investigation of FAOD.

Keywords: active site/biosensor/docking/fructosyl amine oxidase/substrate specificity

Received February 16, 2007; revised August 3, 2007; accepted August 3, 2007.


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