Selective steroid oxyfunctionalisation by CYP154C5, a bacterial cytochrome P450

Abstract

BackgroundCytochrome P450 monooxygenases – able to regio- and stereoselectively hydroxylate non-activated carbon atoms – are important enzymes for the synthesis of valuable intermediates in the production of steroid hormones in the pharmaceutical industry. However, up to now only a few bacterial enzymes able to hydroxylate steroids have been reported. CYP154C5 from Nocardia farcinica IFM 10152, a bacterial P450 monooxygenase, was previously shown to convert testosterone to 16α-hydroxytestosterone. Since the hydroxylation at 16α-position is of special interest for the pharmaceutical industry, we have studied this enzyme in more detail to investigate its activity and selectivity in bioconversions of further steroids.ResultsCYP154C5 was coexpressed in Escherichia coli together with putidaredoxin and putidaredoxin reductase from Pseudomonas putida as redox partners for electron transfer and applied in bioconversions of various pregnanes and androstanes [pregnenolone (1), dehydroepiandrosterone (2), progesterone (3), androstenedione (4), testosterone (5) and nandrolone (6)]. Structure elucidation of the formed products revealed an exclusive regio- and stereoselectivity of CYP154C5, always yielding the corresponding 16α-hydroxylated steroids. Application of whole cells expressing the three components, P450, Pdx and PdR, in steroid biotransformations resulted in significantly higher conversions and total turnover numbers (TTN) compared to reactions using cell-free extracts. Additionally, considerably higher substrate loads (up to 15 mM) were tolerated by the whole-cell system. Furthermore, turnover numbers (TON) were determined for the six different steroids using whole cells. Thus, testosterone was found to be the worst substrate with a TON of only 0.8 μmol substrate consumed min-1 μmol-1 CYP154C5, while progesterone and pregnenolone were converted the fastest resulting in TON of 3.3 μmol substrate consumed min-1 μmol-1 CYP154C5.ConclusionCYP154C5 from N. farcinica constitutes a promising catalyst due to its high regio- and stereoselectivity in the hydroxylation of different steroids as well as its efficient expression in E. coli at high yields. Using this enzyme, 16α-hydroxylated steroids, which are important precursors for the synthesis of high value steroidal drugs in the pharmaceutical industry, can be selectively produced on preparative scale with TTN (μmol substrate consumed μmol-1 CYP154C5) exceeding 2000.

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

“…Its formation was shown to be catalyzed by membrane‐bound cytochrome P450s in human and rat liver microsomes;50 beyond that, the hydroxylation of testosterone at the 16α‐position is an important step in the formation of estriol in late pregnancy 51, 52. Soluble P450s of Gram‐positive bacteria, such as CYP154C3 from Streptomyces griseus , P450 BM3 (CYP102A1, variant M01 A82W S72I) from Bacillus megaterium , and CYP145C5 from Nocardia farcinica , were reported to selectively hydroxylate testosterone and related steroids at the 16α‐position 8, 53, 54. Crude preparations of the latter enzyme were particularly efficient (as an isolated P450) and achieved TTNs of 500 to 2300 for this reaction 54.…”

“…Soluble P450s of Gram‐positive bacteria, such as CYP154C3 from Streptomyces griseus , P450 BM3 (CYP102A1, variant M01 A82W S72I) from Bacillus megaterium , and CYP145C5 from Nocardia farcinica , were reported to selectively hydroxylate testosterone and related steroids at the 16α‐position 8, 53, 54. Crude preparations of the latter enzyme were particularly efficient (as an isolated P450) and achieved TTNs of 500 to 2300 for this reaction 54. We achieved a TTN above 7000 for testosterone oxidation, yet, in contrast to P450s, Cgl UPO requires neither reduced nicotinamide cofactors nor any regenerating system—hydrogen peroxide alone was sufficient for activation and function.…”

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

“…The CYP154C5 enzyme of N. francinica has been shown to hydroxylate six steroids, testosterone (compound 1), pregnenolone (compound 15), dehydroepiandrosterone (compound 11), progesterone (compound 3), ⌬ 4 -androstene-3,17-dione (compound 6), and nandrolone, at the D ring in the 16␣ position, similar to CYP154C3 (14,22). These two CYP154C enzymes are phyloge- netically close to each other (both show 66% identity in the amino acid sequence).…”

“…Interestingly, the substrate specificity of CYP154C3 seems to be different from that of CYP154C5. Progesterone is preferable to testosterone as the substrate for CYP154C5 (22). In contrast, CYP154C3 hydroxylates both progesterone and testosterone with similar efficiency.…”

“…In contrast, CYP154C5 of N. francinica has been shown to hydroxylate testosterone at the 16␣ position of the D ring by a bioconversion experiment (13). Very recently Bracco et al showed that this enzyme can also hydroxylate five other steroids (pregnenolone, dehydroepiandrosterone, progesterone, ⌬ 4 -androstene-3,17-dione, and nandrolone) at the 16␣ position of the D ring (22). It is also noteworthy that in 1957, Streptomyces roseochromogenus was reported to hydroxylate two steroids (9␣-fluorohydrocortisone and 9␣-fluoroprednisolone) at the 16␣ position of the D ring (23), suggesting that S. roseochromogenus has a P450 that catalyzes a reaction similar to that of CYP154C5 of N. francinica.…”