Substrate Hunting for the Myxobacterial CYP260A1 Revealed New 1α‐Hydroxylated Products from C‐19 Steroids

Abstract

Cytochromes P450 catalyze a variety of synthetically useful reactions. However, it is difficult to determine their physiological or artificial functions when a plethora of orphan P450 systems are present in a genome. CYP260A1 from Sorangium cellulosum So ce56 is a new member among the 21 available P450s in the strain. To identify putative substrates for CYP260A1 we used high-throughput screening of a compound library (ca. 17,000 ligands). Structural analogues of the type I hits were searched for biotechnologically relevant compounds, and this led us to select C-19 steroids as potential substrates. We identified efficient surrogate redox partners for CYP260A1, and an Escherichia coli-based whole-cell biocatalyst system was developed to convert testosterone, androstenedione, and their derivatives methyltestosterone and 11-oxoandrostenedione. A detailed (1) H and (13) C NMR characterization of the product(s) from C-19 steroids revealed that CYP260A1 is the very first 1α-steroid hydroxylase.

Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3

“…Androstenedione ( 1 ) was the most common precursor of male and female sex hormones. The hydroxylated modifications of androstenedione ( 1 ), including hydroxylation at positions 1 α , 7 α , 9 α , 11 α , 14 α , 15 α , 16 α , 1 β , 6 β , 15 β and 17 β had been demonstrated previously16, 26. There were four P450 enzymes displaying 1 α -hydroxylated activity towards androstenedione ( 1 )16, 26.…”

“…The chemical synthesis of 1 α -hydroxylated product was still a very challenging process, requiring multi-step reactions and leading to very low yield of 1 α -hydroxyandrostenedione 26 . Biocatalyst-mediated hydroxylation of androstenedione ( 1 ) was thus regarded as an alternative.…”

“…Biocatalyst-mediated hydroxylation of androstenedione ( 1 ) was thus regarded as an alternative. Up to date, myxobacterial CYP260A1 26 and other three BM3 variants 16 were reported to have the hydroxylating capacity of androstenedione ( 1 ) at C-1 position. However, the yield of the 1 α -hydroxylated product catalyzed by these P450 enzymes was not mentioned in these articles16, 26.…”

“…Up to date, myxobacterial CYP260A1 26 and other three BM3 variants 16 were reported to have the hydroxylating capacity of androstenedione ( 1 ) at C-1 position. However, the yield of the 1 α -hydroxylated product catalyzed by these P450 enzymes was not mentioned in these articles16, 26. Therefore, to investigate whether C-1 α hydroxylation catalyzed by BM3 mutant 139-3 could be industrially used, an optimization of catalyzing conditions was performed.…”

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

“…However, few CYPs are known to display such characteristics in the presence of specific chemical compounds. The characterization or synthetic application of CYPs has often been mediated through one or more surrogate redox partners either in an isolated form or after artificial fusion with the CYP complex, due to the difficulty in obtaining native redox partners . It is generally believed that the choice of surrogate partners or their mode of action does not affect the type and selectivity of the reactions catalyzed by CYPs .…”