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  • Title: Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β-Hydroxylation of Lithocholic Acid.
    Author: Grobe S, Badenhorst CPS, Bayer T, Hamnevik E, Wu S, Grathwol CW, Link A, Koban S, Brundiek H, Großjohann B, Bornscheuer UT.
    Journal: Angew Chem Int Ed Engl; 2021 Jan 11; 60(2):753-757. PubMed ID: 33085147.
    Abstract:
    We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and regioselective 7β-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β-position but LCA is exclusively hydroxylated at the 6β-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.
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