Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7ß-Hydroxylation of Lithocholic Acid.
Angew Chem Int Ed Engl
; 60(2): 753-757, 2021 01 11.
Article
em En
| MEDLINE
| ID: mdl-33085147
ABSTRACT
We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and regioselective 7ß-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7ß-position but LCA is exclusively hydroxylated at the 6ß-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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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Ácido Ursodesoxicólico
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Sistema Enzimático do Citocromo P-450
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article