Grogan, Gideon James orcid.org/0000-0003-1383-7056, Rowlinson, Benjamin, Sharma, Mahima orcid.org/0000-0003-3960-2212 et al. (5 more authors) (2021) Substrate anchoring and flexibility reduction in CYP153AM.aq leads to highly improved activity towards octanoic acid. ACS Catalysis. pp. 3182-3189. ISSN 2155-5435
Abstract
Cytochrome P450 CYP153AM.aq from Marinobacter aquaeolei serves as a model enzyme for the terminal (ω-) hy-droxylation of medium- to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database, crystal structures of CYP153M.aq. and a homolog CYP153AP.sp and site-directed saturation mutagenesis, to create a variant that ω-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding, indicated by a 25-fold reduced Km compared to the wild type. Hence, we further investigated the variant computationally using MD simulations to gain deeper insights into the dynamics of the protein. We found the tunnel modifications and the two loop regions showing greatly reduced flexibility in the new variant were the main features responsible for stabilizing the ligand in the active site and enhancing catalytic efficiency. Additionally, we showed that a previously known fatty acid anchor (Q129R) interacts significantly with the ligand to hold it in the reactive position, thereby boosting the activity of the variant M.aq RLT towards octanoic acid. The study retrospectively demonstrates the drastic effects of substrate stabilization and simultaneously the impact of enzyme flexibility. These results could guide future engineering of enzymes with deeply buried active sites to increase or even establish new substrate activities.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2021 The Authors. Published by American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Chemistry (York) The University of York > Faculty of Sciences (York) > Biology (York) |
Funding Information: | Funder Grant number BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL) BB/T017805/1 |
Depositing User: | Pure (York) |
Date Deposited: | 03 Mar 2021 12:40 |
Last Modified: | 08 Feb 2025 00:41 |
Published Version: | https://doi.org/10.1021/acscatal.0c05193 |
Status: | Published online |
Refereed: | Yes |
Identification Number: | 10.1021/acscatal.0c05193 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:171768 |