title: Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase.
abstract: 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 (kcat/KmNADPH = 760 mm-1 min-1), as compared with the wild-type enzyme (kcat/KmNADPH = 133330 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 = 155000 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona), Spain.
authors.forename: Albert
authors.initials: A
authors.lastname: Rosell
authors.forename: Eva
authors.initials: E
authors.lastname: Valencia
authors.forename: Wendy F
authors.initials: WF
authors.lastname: Ochoa
authors.forename: Ignacio
authors.initials: I
authors.lastname: Fita
authors.forename: Xavier
authors.initials: X
authors.lastname: Parés
authors.forename: Jaume
authors.initials: J
authors.lastname: Farrés
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D003903:Deuterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D011898:Ranidae; D013997:Time Factors
chemical_list: D009249:NADP; D003903:Deuterium; C475662:alcohol dehydrogenase 8; D000426:Alcohol Dehydrogenase; D008239:Lysine