Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7?-Hydroxylation of Lithocholic Acid.

ABSTRACT: We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and regioselective 7?-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7?-position but LCA is exclusively hydroxylated at the 6?-position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity-determining residues. Alanine scanning identified S240A as a UDCA-producing variant. A synthetic "small but smart" library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio- and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10-fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA.

SUBMITTER: Grobe S 

PROVIDER: S-EPMC7839452 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature