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Substrate anchoring and flexibility reduction in leads to highly improved efficiency toward octanoic acid

DOI: 10.1021/acscatal.0c05193 DOI Help

Authors: Lea R. Rapp (University of Stuttgart) , Sérgio M. Marques (Masaryk University; St. Anne’s University Hospital Brno) , Erna Zukic (University of York) , Benjamin Rowlinson (University of York) , Mahima Sharma (University of York) , Gideon Grogan (University of York) , Jiri Damborsky (Masaryk University; St. Anne’s University) , Bernhard Hauer (University of Stuttgart)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: February 2021
Diamond Proposal Number(s): 9948

Open Access Open Access

Abstract: Cytochrome P450 from Marinobacter aquaeolei serves as a model enzyme for the terminal (ω-) 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 and a homologue CYP153AP.sp. Applying these focused mutagenesis strategies and site-directed saturation mutagenesis, we created a variant that ω-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding (25-fold reduced Km 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.

Journal Keywords: biocatalysis; enzyme engineering; molecular dynamics; computational chemistry; cytochrome P450

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength)


Discipline Tags:

Catalysis Life Sciences & Biotech Structural biology Chemistry Biochemistry

Technical Tags:

Diffraction Macromolecular Crystallography (MX)