Int. J. Mol. Sci. 2012, 13(11), 14149-14157; doi:10.3390/ijms131114149
Article

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

1email, 1email, 2email, 1email, 1email and 1,2,* email
Received: 20 September 2012; in revised form: 23 October 2012 / Accepted: 26 October 2012 / Published: 2 November 2012
(This article belongs to the Special Issue Flavins)
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Abstract: Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.
Keywords: glucose oxidase; glucose dehydrogenase; FAD; oxygen; site directed mutagenesis
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Horaguchi, Y.; Saito, S.; Kojima, K.; Tsugawa, W.; Ferri, S.; Sode, K. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity. Int. J. Mol. Sci. 2012, 13, 14149-14157.

AMA Style

Horaguchi Y, Saito S, Kojima K, Tsugawa W, Ferri S, Sode K. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity. International Journal of Molecular Sciences. 2012; 13(11):14149-14157.

Chicago/Turabian Style

Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji. 2012. "Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity." Int. J. Mol. Sci. 13, no. 11: 14149-14157.


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