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J. Biol. Chem., Vol. 278, Issue 42, 40573-40580, October 17, 2003

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Complete Reversal of Coenzyme Specificity by Concerted Mutation of Three Consecutive Residues in Alcohol Dehydrogenase^*

Albert Rosell , Eva Valencia , Wendy F. Ochoa , Ignacio Fita , Xavier Parés  and Jaume Farrés  ¶

From the Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona), Spain and the IBMB-Consejo Superior de Investigaciones Científicas, Jordi-Girona 18-26, E-08034 Barcelona, Spain

Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP⁺ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly²²³-Thr²²⁴-His²²⁵, and the highly conserved Leu²⁰⁰ and Lys²²⁸. To investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. Computer-assisted modeling of the docked coenzymes was also performed with the mutant enzymes and compared with the wild-type crystallographic binary complex. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (k_cat/K_mNADPH = 760 mM^-1 min^-1), as compared with the wild-type enzyme (k_cat/K_mNADPH = 133,330 mM^-1 min^-1). This was associated with a lower binding affinity for NADP⁺ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (k_cat/K_mNADH = 155,000 mM^-1 min^-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.

Received for publication, July 10, 2003 , and in revised form, August 4, 2003.

^* This work was supported by grants from the Dirección General de Investigación Científica: BMC2000-0132 (to J. F.), BMC2002-02659 (to X. P.), and BIO2002-04419-C02-01 (to I. F.). 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 Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona), Spain. Tel.: 34-93-5812557; Fax: 34-93-5811264; E-mail: jaume.farres{at}uab.es.

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