CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

Abstract

The guidelines of the Food and Drug Administration and International Conference on Harmonization have highlighted the importance of drug metabolites in clinical trials. As a result, an authentic source for their production is of great interest, both for their potential application as analytical standards and for required toxicological testing. Since we have previously shown promising biotechnological potential of cytochromes P450 from the soil bacterium Sorangium cellulosum So ce56, herein we investigated the CYP267 family and its application for the conversion of commercially available drugs including nonsteroidal anti-inflammatory, antitumor, and antihypotensive drugs. The CYP267 family, especially CYP267B1, revealed the interesting ability to convert a broad range of substrates. We established substrate-dependent extraction protocols and also optimized the reaction conditions for the in vitro experiments and Escherichia coli-based whole-cell bioconversions. We were able to detect activity of CYP267A1 toward seven out of 22 drugs and the ability of CYP267B1 to convert 14 out of 22 drugs. Moderate to high conversions (up to 85% yield) were observed in our established whole-cell system using CYP267B1 and expressing the autologous redox partners, ferredoxin 8 and ferredoxin-NADP + reductase B. With our existing setup, we present a system capable of producing reasonable quantities of the human drug metabolites 49-hydroxydiclofenac, 2-hydroxyibuprofen, and omeprazole sulfone. Due to the great potential of converting a broad range of substrates, wild-type CYP267B1 offers a wide scope for the screening of further substrates, which will draw further attention to future biotechnological usage of CYP267B1 from S. cellulosum So ce56.

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

“…Protein purity was evaluated by SDS-PAGE ( Supplementary Figure 1) and the purified enzyme showed its typical spectral properties as reported previously [26].…”

“…The whole-cell reaction for the conversion of flavanone was performed as described previously [26]. The E. coli BL21 (DE3) cells were transformed with two plasmids, one encoding CYP267B1 (pET22b_CYP267B1) and one encoding the autologous redox partners from S. cellulosum So ce56, FdR_B (Ferredoxin-NADP + reductase) and Fdx8 (Ferredoxin 8) (pCDF_F8B).…”

“…The in vitro conversion of flavanone by CYP267B1 resulted in one major product (P1, 15%) and some undefined side products (P2-P5) as detected by HPLC analysis (Figure 2A). In order to identify the products by NMR analysis, the previously established E. coli-based whole-cell system was used to scale-up the flavanone conversion [26]. In the applied in vivo system CYP267B1 was supported by an autologous redox partner pair, the FdR_B Figure 2B).…”

“…Without the need for any protein engineering, the enzyme was shown to readily accept different classes of substrates, from small carotenoid-derived aroma compounds or sesquiterpenes [22,23] to sterically challenging molecules like epothilone D [24]. CYP267B1 was also shown to oxidize several structurally diverse drugs, such as diclofenac, ibuprofen, oxymetazoline or repaglinide, as well as antidepressant or antipsychotic drugs like amitriptyline, chlorpromazine, imipramine or promethazine [25,26]. Thus, the wild type CYP267B1 shows high catalytic variability, catalyzing not only aliphatic, allylic and aromatic hydroxylations, but also sulfoxidation or epoxidation [22,26].…”

“…CYP267B1 was also shown to oxidize several structurally diverse drugs, such as diclofenac, ibuprofen, oxymetazoline or repaglinide, as well as antidepressant or antipsychotic drugs like amitriptyline, chlorpromazine, imipramine or promethazine [25,26]. Thus, the wild type CYP267B1 shows high catalytic variability, catalyzing not only aliphatic, allylic and aromatic hydroxylations, but also sulfoxidation or epoxidation [22,26]. Despite the wealth of biochemical data available for this versatile P450, its catalytic potential towards different substrate classes might not be fully explored and its structural properties have not been investigated yet.…”