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.
Subject(s)
Bacterial Proteins/metabolism , Cytochrome P-450 Enzyme System/metabolism , Ursodeoxycholic Acid/metabolism , Bacterial Proteins/genetics , Binding Sites , Catalytic Domain , Cytochrome P-450 Enzyme System/genetics , Deoxycholic Acid/chemistry , Deoxycholic Acid/metabolism , Hydroxylation , Lithocholic Acid/chemistry , Lithocholic Acid/metabolism , Molecular Docking Simulation , Mutagenesis, Site-Directed , Stereoisomerism , Streptomyces/enzymology , Ursodeoxycholic Acid/chemical synthesis , Ursodeoxycholic Acid/chemistryABSTRACT
OBJECTIVE: Regio- and stereoselective hydroxylation of lithocholic acid (LCA) using CYP107D1 (OleP), a cytochrome P450 monooxygenase from the oleandomycin synthesis pathway of Streptomyces antibioticus. RESULTS: Co-expression of CYP107D1 from S. antibioticus and the reductase/ferredoxin system PdR/PdX from Pseudomonas putida was performed in Escherichia coli whole cells. In vivo hydroxylation of LCA exclusively yielded the 6ß-OH product murideoxycholic acid (MDCA). In resting cells, 19.5% of LCA was converted to MDCA within 24 h, resulting in a space time yield of 0.04 mmol L-1 h-1. NMR spectroscopy confirmed the identity of MDCA as the sole product. CONCLUSIONS: The multifunctional P450 monooxygenase CYP107D1 (OleP) can hydroxylate LCA, forming MDCA as the only product.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Lithocholic Acid/chemistry , Streptomyces antibioticus/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biocatalysis , Cloning, Molecular , Deoxycholic Acid/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , Hydroxylation , Oxidoreductases/genetics , Oxidoreductases/metabolism , Pseudomonas putida/enzymology , Pseudomonas putida/genetics , Streptomyces antibioticus/geneticsABSTRACT
The CYP171 enzyme is known to catalyse a key step in the steroidogenesis of mammals. The substrates progesterone and pregnenolone are first hydroxylated at the C17 position, and this is followed by cleavage of the C17-C20 bond to yield important precursors for glucosteroids and androgens. In this study, we focused on the reaction of the bovine CYP17A1 enzyme with progesterone as a substrate. On the basis of a created homology model, active-site residues were identified and systematically mutated to alanine. In whole-cell biotransformations, the importance of the N202, R239, G297 and E305 residues for substrate conversion was confirmed. Additionally, mutation of the L206, V366 and V483 residues enhanced the formation of the 16α-hydroxyprogesterone side product up to 40 % of the total product formation. Furthermore, residue L105 was found not to be involved in this side activity, which contradicts a previous study with the human enzyme.
