The Role of Substrate-Coenzyme Crosstalk in Determining Turnover Rates in Rhodococcus ruber Alcohol Dehydrogenase

Eight related alcohol dehydrogenases that had been originally isolated by laboratory evolution of ADH-A from Rhodococcus ruber DSM44541 for modified substrate scopes, were together with their parent wild-type, subjected to biochemical characterization of possible activities with a panel of chiral alcohols and pro-chiral ketones. Determinations of rates of catalyzed alcohol oxidations and ketone reductions, and of cofactor release, pointed out to the role of a W295A substitution as being decisive in steering enantioselectivity in the oxidation of arylated 1-methyl substituted alcohols. Molecular dynamics simulations of enzyme-substrate interactions in the Michaelis complexes of wild-type and a Y294F/W295A double mutant could rationalize the experimentally observed shift in enantioselectivity and differences in catalytic activity with 4-phenyl-2-butanol. Finally, we present herein evidence for apparent inter-dependency between substrate/product and the cofactor in the ternary complex, that directly affects the NADH dissociation rates, and thus that this substrate-coenzyme crosstalk plays a direct role in determining the turnover rates.

NATURVETENSKAP Biologi Biokemi och molekylärbiologi hsv//swe
NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology hsv//eng
alcohol dehydrogenase
substrate selectivity
active-site structure
stereoselectivity
molecular dynamics simulations
Biokemi
Biochemistry

Corbella Morató, MarinaUppsala universitet,Biokemi,Kamerlin(Swepub:uu)marco394 (författare)
Kamerlin, Shina C. L.,1981-Uppsala universitet,Biokemi,Kamerlin(Swepub:uu)lynka392 (författare)
Widersten, MikaelUppsala universitet,Biokemi,Widersten(Swepub:uu)mikawide (författare)
Uppsala universitetBiokemi (creator_code:org_t)

Ingår i:ACS Catalysis: American Chemical Society (ACS)10:16, s. 9115-91282155-5435

Av författaren/redakt...: Enugala, Thilak ...; Corbella Morató, ...; Kamerlin, Shina ...; Widersten, Mikae ...

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