Highly selective bile acid hydroxylation by the multifunctional bacterial P450 monooxygenase CYP107D1 (OleP)

Abstract

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 6b-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.

Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate

“…In light of redirecting the epoxidase activity of OleP toward a broader range of molecules as a biocatalyst for the production of new chemicals and drugs [ 10 , 11 ], it is of interest to underline how an additional methyl group at the C13 substituent of the aglycone substrate, as in 6DEB, is sufficient to increases the binding affinity of OleP by one order of magnitude. This observation, together with the structural information herein reported, may provide guidance in the choice of new target molecules and/or in the rational engineering of OleP to redirect its activity towards alternative substrates.…”

“…Among the known macrolide epoxygenases from actinomycetes, OleP presents a unique epoxidation chemistry since it targets a non-activated C-C bond, which is a reaction not easily achievable through conventional chemical synthesis protocols. This makes OleP a valuable target for protein engineering aimed at redirecting its epoxidase reactivity [ 10 , 11 ]. However, many aspects of the mechanism behind its enzymatic reaction are still unclear.…”

Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β‐Hydroxylation of Lithocholic Acid

“…OleP can also hydroxylate testosterone at the positions 6β, 7β, 12β, and 15β [25] . However, bile acids like LCA and deoxycholic acid (DCA) are hydroxylated exclusively at the 6β‐position, forming MDCA (Scheme 1 b) and 3α‐, 6β‐, 12α‐trihydroxy‐5β‐cholan‐24‐oic acid, respectively [26] …”

“…[25] However, bile acids like LCA and deoxycholic acid (DCA) are hydroxylated exclusively at the 6b-position, forming MDCA (Scheme 1 b) and 3a-, 6b-, 12a-trihydroxy-5b-cholan-24-oic acid, respectively. [26] To open up a novel reaction pathway towards UDCA, as no enzyme is known for selective 7b-hydroxylation, and to avoid using whole cell fungi, wherein the action of multiple P450s frequently results in side-product formation, we decided to engineer OleP and create an Escherichia coli (E. coli)-based whole-cell system for the regio-and stereoselective hydroxylation of LCA at the 7b-position to form UDCA (Scheme 1 b).…”

Modifikation der Regioselektivität einer P450‐Monooxygenase ermöglicht die Synthese von Ursodeoxycholsäure durch die 7β‐Hydroxylierung von Lithocholsäure

“…[25] Hingegen werden Gallensäuren wie LCS und Deoxycholsäure (DCS) ausschließlich an der 6b-Position hydroxyliert, wodurch MDCS (Schema 1 b) beziehungsweise 3a-,6b-,12a-Trihydroxy-5b-cholan-24-olsäure gebildet werden. [26] Um einen Reaktionsweg zu UDCS zu erçffnen, da kein Enzym für die selektive 7b-Hydroxylierung bekannt ist, und um die Verwendung von Pilzen zu vermeiden, in welchen mehrere P450-Enzyme Nebenprodukte bilden, haben wir uns dazu entschlossen, OleP durch Protein-Engineering anzupassen, sodass ein Escherichia coli (E. coli)-basiertes Ganzzellsystem für die regio-und stereoselektive Hydroxylierung von LCS zur UDCS entsteht (Schema 1 b).…”