Selective steroid oxyfunctionalisation by CYP154C5, a bacterial cytochrome P450

Area of Science:

• Biotechnology
• Enzymology
• Organic Chemistry

Background:

• Cytochrome P450 monooxygenases are crucial for synthesizing steroid hormone intermediates.
• Few bacterial enzymes capable of steroid hydroxylation have been identified.
• CYP154C5 from Nocardia farcinica was previously shown to hydroxylate testosterone at the 16α-position.

Purpose of the Study:

• To investigate the activity and selectivity of CYP154C5 in the bioconversion of various steroids.
• To explore the potential of CYP154C5 for producing 16α-hydroxylated steroids, which are important pharmaceutical precursors.

Main Methods:

• Coexpression of CYP154C5 with redox partners (putidaredoxin and putidaredoxin reductase) in Escherichia coli.
• Bioconversion of androstanes and pregnanes using whole cells and cell-free extracts.
• Structure elucidation of the hydroxylated steroid products.
• Determination of turnover numbers (TON) and total turnover numbers (TTN).

Main Results:

• CYP154C5 exhibited exclusive regio- and stereoselectivity, yielding 16α-hydroxylated steroids from various substrates.
• Whole-cell biotransformations showed significantly higher conversions and TTN compared to cell-free systems.
• The whole-cell system tolerated higher substrate loads (up to 15 mM).
• Progesterone and pregnenolone were the fastest converted substrates (TON of 3.3 μmol min⁻¹ μmol⁻¹ CYP154C5).

Conclusions:

• CYP154C5 is a promising catalyst for selective steroid hydroxylation due to its high selectivity and efficient expression in E. coli.
• The enzyme facilitates the preparative-scale production of 16α-hydroxylated steroids with TTN exceeding 2000.
• These 16α-hydroxylated steroids are key precursors for high-value steroidal drugs.