The 16α‐Hydroxylation of Progesterone by Cytochrome P450 107X1 from Streptomyces avermitilis

Abstract

Cytochrome P450 enzymes (CYPs or P450s) are ubiquitous heme‐dependent enzymes that catalyze the monooxygenation of non‐activated C−H bonds to modify the structure of the substrate. In this study, we heterologously expressed CYP107X1 from Streptomyces avermitilis and conducted in vitro substrate screening using the alternative redox partners putidaredoxin and putidaredoxin reductase. CYP107X1 catalyzed the 16α‐hydroxylation of progesterone with regio‐ and stereoselectivity. The spectroscopic analyses showed that CYP107X1 bound progesterone with a relatively high Kd value of 65.3±38.9 μM. The Km and kcat values for progesterone were estimated to be 47.7±12.0 μM and 0.30 min−1, respectively. Furthermore, a crystal structure was obtained of CYP107X1 bound with glycerol from the buffer solution. Interestingly, a conserved threonine was replaced with asparagine in CYP107X1, indicating that it may adopt an unnatural proton transfer process and play a crucial role in its catalytic activity.

Structural Insights into the Interaction of Terpenoids with Streptomyces avermitilis CYP107P2

“…Utilizing the beamline 11C-Micro-MX at the Pohang Light Source (PLS, Pohang, Korea), X-ray diffraction data were collected by rotating 1° at a time with a 400 mm crystal-to-detector distance using a Pilatus 3 6M detector. The CYP107P2 structure was determined using molecular replacement in CCP4 MolRep with a search model based on CYP107X1 (PDB ID: 7WEX, 41.6% sequence identity) ( Lin et al ., 2022 ). The structure was determined using COOT with the search model and refined using REFMAC5 ( Emsley and Cowtan, 2004 ).…”

“…the molecular replacement of CYP107X1 from S. avermitilis (PDB ID: 7WEX) (Lin et al, 2022). Excluding the first seven N-terminal residues, and the residues Gly182 and Met183, an electron density map was observed, including the active site of the P450 enzyme (Supplementary Fig.…”

“…P450 PikC from S. venezuelae , which shares approximately 41% sequence similarity with CYP107P2, plays a role in hydroxylating macrolides such as YC-17 and narbomycin ( Sherman et al ., 2006 ). Moreover, CYP107X1 from S. avermitilis , sharing 39% sequence similarity with CYP107P2, demonstrated the potential to hydroxylate progesterone at the 16α-hydroxylation, although it is not its physiological substrate ( Lin et al ., 2022 ). The CYP107 family in Streptomyces typically oxidizes and epoxidizes a wide range of substrates, highlighting a flexible active site capable of accommodating diverse and large substrates ( Padayachee et al ., 2023 ).…”

Structure–Function Analysis of the Biotechnologically Important Cytochrome P450 107 (CYP107) Enzyme Family

“…The 16α-Hydroxylation of progesterone Steroid [38] The CYP107 members studied to-date are soluble proteins, and their general overall structure is comparable to that of bacterial P450s [39,40]. Structural analysis of CYP107 members revealed characteristic P450 folds and conserved motifs in their structures [39,40].…”

“…Over the past few years, the resolved X-ray crystal structures of many CYP107 family members' have been determined. To-date, 44 CYP107 family members' crystal structures Steroid [38] The CYP107 members studied to-date are soluble proteins, and their general overall structure is comparable to that of bacterial P450s [39,40]. Structural analysis of CYP107 members revealed characteristic P450 folds and conserved motifs in their structures [39,40].…”