1. Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β‐Hydroxylation of Lithocholic Acid.
- Author
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Grobe, Sascha, Badenhorst, Christoffel P. S., Bayer, Thomas, Hamnevik, Emil, Wu, Shuke, Grathwol, Christoph W., Link, Andreas, Koban, Sven, Brundiek, Henrike, Großjohann, Beatrice, and Bornscheuer, Uwe T.
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URSODEOXYCHOLIC acid , *WASTE products , *CYTOCHROME P-450 , *MOLECULAR docking , *ACIDS , *AMINO acid residues - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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