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Computational design of an amidase by combining the best electrostatic features of two promiscuous hydrolases

Galmés, Miquel À., Nodling, Alexander R., He, Kaining, Luk, Louis Y. P. ORCID:, Swiderek, Katarzyna and Moliner, Vicent 2022. Computational design of an amidase by combining the best electrostatic features of two promiscuous hydrolases. Chemical Science 13 (17) , pp. 4779-4787. 10.1039/D2SC00778A

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While there has been emerging interest in designing new enzymes to solve practical challenges, computer-based options to redesign catalytically active proteins are rather limited. Here, a rational QM/MM molecular dynamics strategy based on combining the best electrostatic properties of enzymes with activity in a common reaction is presented. The computational protocol has been applied to the re-design of the protein scaffold of an existing promiscuous esterase from Bacillus subtilis Bs2 to enhance its secondary amidase activity. After the alignment of Bs2 with a non-homologous amidase Candida antarctica lipase B (CALB) within rotation quaternions, a relevant spatial aspartate residue of the latter was transferred to the former as a means to favor the electrostatics of transition state formation, where a clear separation of charges takes place. Deep computational insights, however, revealed a significant conformational change caused by the amino acid replacement, provoking a shift in the pKa of the inserted aspartate and counteracting the anticipated catalytic effect. This prediction was experimentally confirmed with a 1.3-fold increase in activity. The good agreement between theoretical and experimental results, as well as the linear correlation between the electrostatic properties and the activation energy barriers, suggest that the presented computational-based investigation can transform in an enzyme engineering approach.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Additional Information: This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence
Publisher: Royal Society of Chemistry
ISSN: 2041-6520
Funders: BBSRC, Leverhulme Trust and Royal Society
Date of First Compliant Deposit: 12 April 2022
Date of Acceptance: 14 February 2022
Last Modified: 05 May 2023 11:21

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