Probing stereoselectivity and pro-chirality of hydride transfer during short-chain alcohol dehydrogenase activity: a combined quantitative 2H NMR and computational approach.
Different members of the alcohol oxidoreductase family can transfer the hydride of NAD(P)H to either the re- or the si-face of the substrate. The enantioselectivity of transfer is very variable, even for a range of substrates reduced by the same enzyme. Exploiting quantitative isotopic (2)H NMR to measure the transfer of (2)H from NAD(P)(2)H to ethanol, a range of enantiomeric excess between 0.38 and 0.98, depending on the origin of the enzyme and the nature of the cofactor, has been determined. Critically, in no case was only (R)-[1-(2)H]ethanol or (S)-[1-(2)H]ethanol obtained. By calculating the relative energies of the active site models for hydride transfer to the re- or si-face of short-chain aldehydes by alcohol dehydrogenase from Saccharomyces cerevisiae and Lactobacillus brevis, it is shown that the differences in the energy of the systems when the substrate is positioned with the alkyl group in one or the other pocket of the active site could play a role in determining stereoselectivity. These experiments help to provide insight into structural features that influence the potential catalytic flexibility of different alcohol dehydrogenase activities.
Kwiecień
Renata A
RA
Unit for Interdisciplinary Chemistry: Synthesis, Analysis, Modeling, UMR CNRS6230, CNRS, University of Nantes, 2 rue de la Houssinière, Nantes, France.
Ayadi
Farouk
F
Nemmaoui
Youssef
Y
Silvestre
Virginie
V
Zhang
Ben-Li
BL
Robins
Richard J
RJ
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D044926:Butyryl-CoA Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry; D020134:Catalytic Domain; D000431:Ethanol / Q000378:metabolism; D007700:Kinetics; D052196:Levilactobacillus brevis / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D000596:Amino Acids; D001426:Bacterial Proteins; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D000431:Ethanol; D009249:NADP; D044926:Butyryl-CoA Dehydrogenase