Laboratory evolution of proteins has opened up new possibilities for the emergence of novel functional properties and the development of new biocatalysts. Mutagenesis events can unveil new catalytic properties as a consequence of the improvement of hidden promiscuous activities. Chiral alcohols are some of the most common building blocks used in the pharmaceutical industry, and alcohol dehydrogenases are valuable biocatalysts used for the production of optically pure chiral alcohols. Engineered variants of alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 were shown to revert the 270-fold preference of the wild-type enzyme towards the (S)-enantiomer of 1-phenylethanol into a 7.4-fold enantiopreference for the (R)-1-phenylethanol. In the present work we selected a number of ADH-A variants engineered for different purposes and functions with the objective of elucidating intrinsic promiscuities in substrate selectivity towards a spectrum of potential alcohol and ketone substrates, as well as assessing their biocatalytic potential of resolved alcohol oxidation and stereoselective ketone reduction. We characterized the enzymes with respect to their activities during the steady state phase of the catalyzed reaction with emphasis on efficiencies in catalytic turnover and stereoselectivities. Computational modeling through molecular dynamics simulations was used together to explore different putative substrate binding modes, as well as to rationalize the impact of such modifications to the observed ADH-A stereoselectivity. The wild-type enzyme was found to be, with some exceptions, the enzyme variant with widest, generalist type, substrate scope. However, our results show that subtle changes in the volume and shape of the active site lead to alterations in the conformational ensembles for the two enantiomers that reverse the enantioselectivity of ADH-A.

Computationally exploring substrate promiscuity in in-vitro evolved alcohol dehydrogenases

ACS Spring Meeting 2021

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