title: Enhancing the efficiency and regioselectivity of P450 oxidation catalysts by unnatural amino acid mutagenesis.
abstract: The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active-site positions of a substrate-promiscuous CYP102A1 variant. The resulting "uP450s" were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small-molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para-acetyl-Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp(3))-H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity-enhancing effect of active-site substitutions involving the unnatural amino acid para-amino-Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34,650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts.
authors.affiliation: Department of Chemistry, University of Rochester, Hutchison Hall, Rochester, NY 14620 (USA).
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Kolev
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Zaengle
authors.forename: Rajesh
authors.initials: R
authors.lastname: Ravikumar
authors.forename: Rudi
authors.initials: R
authors.lastname: Fasan
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation; D007052:Ibuprofen / Q000737:chemistry / Q000378:metabolism; D016296:Mutagenesis; D010084:Oxidation-Reduction; D000081223:Polycyclic Sesquiterpenes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D000081223:Polycyclic Sesquiterpenes; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D003577:Cytochrome P-450 Enzyme System; C050302:nootkatone; D007052:Ibuprofen
keywords: cytochromes; oxidation; protein engineering; terpenes; unnatural amino acids