The Role of Substrate-Coenzyme Crosstalk in Determining Turnover Rates in Rhodococcus ruber Alcohol Dehydrogenase
| Autor: | Thilak Reddy Enugala, Mikael Widersten, Shina Caroline Lynn Kamerlin, Marina Corbella Morató |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
biology
010405 organic chemistry Chemistry alcohol dehydrogenase Biochemistry and Molecular Biology Rhodococcus ruber Alcohol General Chemistry molecular dynamics simulations 010402 general chemistry stereoselectivity 01 natural sciences substrate selectivity Catalysis Cofactor 0104 chemical sciences Crosstalk (biology) chemistry.chemical_compound active-site structure Biochemistry biology.protein bacteria Biokemi och molekylärbiologi Alcohol dehydrogenase |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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