5O8H

Crystal structure of R. ruber ADH-A, mutant Y294F, W295A, F43H, H39Y

Summary for 5O8H

• Entry DOI: 10.2210/pdb5o8h/pdb
• Descriptor: Alcohol dehydrogenase, ZINC ION, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, ... (6 entities in total)
• Functional Keywords: alcohol dehydrogenase mutant variant, nadh-dependent, zn2+-dependent, rossmann fold, oxidoreductase
• Biological source: Rhodococcus sp. M8
• Total number of polymer chains: 4
• Total formula weight: 147286.81

Authors

Dobritzsch, D.,Maurer, D.,Hamnevik, E.,Reddy Enugala, T.,Widersten, M. (deposition date: 2017-06-13, release date: 2017-10-11, Last modification date: 2024-01-17)

Primary citation

Hamnevik, E.,Enugala, T.R.,Maurer, D.,Ntuku, S.,Oliveira, A.,Dobritzsch, D.,Widersten, M.
Relaxation of nonproductive binding and increased rate of coenzyme release in an alcohol dehydrogenase increases turnover with a nonpreferred alcohol enantiomer.
FEBS J., 284:3895-3914, 2017

PubMed Abstract: Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (a) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (b) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate-binding modes.

PubMed: 28963762
DOI: 10.1111/febs.14279

Experimental method

X-RAY DIFFRACTION (1.96 Å)