Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β‐Hydroxylation of Lithocholic Acid
| Autor: | Sascha Grobe, Thomas Bayer, Emil Hamnevik, Sven Koban, Uwe T. Bornscheuer, Shuke Wu, Beatrice Großjohann, Christoph W. Grathwol, Christoffel P. S. Badenhorst, Andreas Link, Henrike Brundiek |
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| Rok vydání: | 2020 |
| Předmět: |
cytochrome P450 monooxygenase
Lithocholic acid Stereochemistry 7β-hydroxylation Hydroxylation 010402 general chemistry 01 natural sciences Catalysis chemistry.chemical_compound Bacterial Proteins Cytochrome P-450 Enzyme System Catalytic Domain medicine Protein Engineering | Very Important Paper Binding Sites biology 010405 organic chemistry Communication Ursodeoxycholic Acid Cytochrome P450 Regioselectivity protein engineering Stereoisomerism General Chemistry Monooxygenase Alanine scanning Communications Streptomyces Ursodeoxycholic acid 0104 chemical sciences Molecular Docking Simulation lithocholic acid chemistry Mutagenesis Site-Directed biology.protein Stereoselectivity Deoxycholic Acid medicine.drug |
| Zdroj: | Angewandte Chemie (International Ed. in English) |
| ISSN: | 1521-3773 1433-7851 |
| Popis: | 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. We report engineering of a P450 monooxygenase for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid to produce ursodeoxycholic acid (UDCA). Structural and 3DM analysis, and molecular docking, identified selectivity‐influencing residues. A “small but smart” mutant library was then screened with a selective colorimetric assay. The best mutant has nearly perfect regio‐ and stereoselectivity, enabling a new route for UDCA synthesis. |
| Databáze: | OpenAIRE |
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