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.
Highlights
Cytochrome 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
P450 monooxygenases are applied in the pharmaceutical industry for the regio- and stereoselective hydroxylation of steroid compounds – a reaction that can be hardly achieved by chemical methods – to form highly valuable steroid hormones like glucocorticoids, mineralocorticoids and sexual hormones [2,4]
It was shown before that putidaredoxin (Pdx) and putidaredoxin reductase (PdR) from the P450cam system of Pseudomonas putida [11,13] as well as the reductase domain of P450RhF (CYP116B2) from Rhodococcus sp
Summary
Cytochrome 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. Cytochrome P450 enzymes (CYPs) form a huge superfamily of heme-containing monooxygenases present in all domains of life, where they play important roles in the detoxification of xenobiotics, drug metabolism, the assimilation of carbon sources and the formation of secondary metabolites [1] They catalyze a number of chemically diverse reactions like hydroxylations, epoxidations, dealkylations and dehalogenations [2,3], making them very attractive catalysts for application in the synthesis of fine chemicals and pharmaceuticals. Agematu et al examined a library consisting of 213 different bacterial CYPs for their ability to convert testosterone [11] Of these 213 enzymes only 24 turned out to be active on this steroid substrate, exhibiting different regio- and stereoselectivities. NCIMB 9784 [14] can be applied as surrogate electron transfer partners required for CYP154C5 activity
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