Substrate Anchoring and Flexibility Reduction in CYP153A(M.aq) Leads to Highly Improved Efficiency toward Octanoic Acid

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Publikace nespadá pod Ekonomicko-správní fakultu, ale pod Přírodovědeckou fakultu. Oficiální stránka publikace je na webu muni.cz.
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RAPP Lea R. MARQUES Sérgio Manuel ZUKIC Erna ROWLINSON Benjamin SHARMA Mahima GROGAN Gideon DAMBORSKÝ Jiří HAUER Bernhard

Rok publikování 2021
Druh Článek v odborném periodiku
Časopis / Zdroj ACS Catalysis
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
www https://pubs.acs.org/doi/10.1021/acscatal.0c05193
Doi http://dx.doi.org/10.1021/acscatal.0c05193
Klíčová slova biocatalysis; enzyme engineering; molecular dynamics; computational chemistry; cytochrome P450
Popis Cytochrome P450 CYP153A(M.aq) from Marinobacter aquaeolei serves as a model enzyme for the terminal (omega-) hydroxylation 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 and crystal structures of CYP153A(M.aq) and a homologue CYP153A(P.sp). Applying these focused mutagenesis strategies and site-directed saturation mutagenesis, we created a variant that omega-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding (25-fold reduced K-m compared to the wild type). We then used molecular dynamics 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 engineered variant were the main features responsible for stabilizing the enzyme-substrate complex and enhancing the 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.aqRLT toward octanoic acid. The study demonstrates the significant effects of both substrate stabilization and the impact of enzyme flexibility on catalytic efficiency. These results could guide the future engineering of enzymes with deeply buried active sites to increase or even establish activities toward yet unknown types of substrates.
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