Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase.
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.
Rosell
Albert
A
Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona), Spain.
Valencia
Eva
E
Ochoa
Wendy F
WF
Fita
Ignacio
I
Parés
Xavier
X
Farrés
Jaume
J
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
D009249:NADP; D003903:Deuterium; C475662:alcohol dehydrogenase 8; D000426:Alcohol Dehydrogenase; D008239:Lysine