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J. Biol. Chem., Vol. 280, Issue 11, 10340-10349, March 18, 2005

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Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability by the Introduction of Structural Zinc^*

Seiya Watanabe¶, Tsutomu Kodaki¶, and Keisuke Makino¶||

From the Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, International Innovation Center, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-8501, and ^¶CREST, JST (Japan Science and Technology Agency), Gokasyo, Uji, Kyoto 611-0011, Japan

Pichia stipitis NAD⁺-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD⁺ to NADP⁺ and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp²⁰⁷, Ile²⁰⁸, Phe²⁰⁹, and Asn²¹¹ in the discrimination between NAD⁺ and NADP⁺. Single mutants (D207A, I208R, F209S, and N211R) improved 548-fold in catalytic efficiency (k_cat/K_m) with NADP⁺ compared with the wild type but retained substantial activity with NAD⁺. The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k_cat/K_m with NADP⁺, but they still preferred NAD⁺ to NADP⁺. The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k_cat/K_m with NADP⁺ than the wild-type enzyme, reaching values comparable with k_cat/K_m with NAD⁺ of the wild-type enzyme. Because most NADP⁺-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP⁺.

Received for publication, August 17, 2004 , and in revised form, December 27, 2004.

^* This work was supported in part by the Center of Excellence (COE) program of the "Establishment of COE on Sustainable-Energy System," grant-in-aid for Scientific Research, and Grants for Regional Science and Technology Promotion from the Ministry of Education, Science, Sports, and Culture, Japan. This work was also supported by CREST of Japan Science and Technology Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011 Japan. Tel.: 81-774-38-3517; Fax: 81-774-38-3524; E-mail: kmak{at}iae.kyoto-u.ac.jp.

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