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Title

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

AuthorsRosell, Albert; Valencia, Eva; Ochoa, Wendy F.; Fita, Ignacio ; Parés, Xavier; Farrés, Jaume
Issue Date17-Oct-2003
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationJournal of Biological Chemistry 278(42): 40573-40580 (2003)
AbstractGastric 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 Gly 223-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 (kcat/K mNADPH = 760 mM-1 min-1), as compared with the wild-type enzyme (kcat/KmNADPH = 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 (kcat/KmNADH = 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.
Publisher version (URL)http://dx.doi.org/10.1074/jbc.M307384200
URIhttp://hdl.handle.net/10261/111051
DOI10.1074/jbc.M307384200
Identifiersdoi: 10.1074/jbc.M307384200
issn: 0021-9258
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