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J. Biol. Chem., Vol. 280, Issue 25, 24104-24112, June 24, 2005

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Molecular Conversion of NAD Kinase to NADH Kinase through Single Amino Acid Residue Substitution^*

Shigetarou Mori, Shigeyuki Kawai, Feng Shi, Bunzo Mikami, and Kousaku Murata¶

From the Department of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan and the Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

NAD kinase phosphorylates NAD⁺ to form NADP⁺ and is strictly specific to NAD⁺, whereas NADH kinase phosphorylates both NAD⁺ and NADH, thereby showing relaxed substrate specificity. Based on their primary and tertiary structures, the difference in the substrate specificities between NAD and NADH kinases was proposed to be caused by one aligned residue: Gly or polar amino acid (Gln or Thr) in five NADH kinases and a charged amino acid (Arg) in two NAD kinases. The substitution of Arg with Gly in the two NAD kinases relaxed the substrate specificity (i.e. converted the NAD kinases to NADH kinases). The substitution of Arg in one NAD kinase with polar amino acids also relaxed the substrate specificity, whereas substitution with charged and hydrophobic amino acids did not show a similar result. In contrast, the substitution of Gly with Arg in one NADH kinase failed to convert it to NAD kinase. These results suggest that a charged or hydrophobic amino acid residue in the position of interest is crucial for strict specificity of NAD kinases to NAD⁺, whereas Gly or polar amino acid residue is not the sole determinant for the relaxed substrate specificity of NADH kinases. The significance of the conservation of the residue at the position in 207 NAD kinase homologues is also discussed.

Received for publication, March 7, 2005 , and in revised form, April 25, 2005.

^* This work was supported in part by Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-in-aid 15780212 and by the Program for Promotion of Basic Research Activities for Innovative Biosciences. 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: Dept. of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan. Tel.: 81-774-38-3766; Fax: 81-774-38-3767; E-mail: kmurata{at}kais.kyoto-u.ac.jp.

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