Probing the Catalytic Promiscuity and Enhancing the Efficiency of CYP725A4 with a Taxadiene Substrate Mimic
利用紫杉二烯底物模拟物探究 CYP725A4 的催化多功能性并提升其催化效率
- 关键词：
- 来源：
- ACS Catalysis
- 类型：
- 学术文献
- 语种：
- 英语
- 原文发布日期：
- 2026-08-24
- 摘要：
- The catalytic promiscuity of cytochrome P450 CYP725A4 severely impedes paclitaxel biosynthesis, as it generates taxadien-5α-ol (T-5α-ol) alongside numerous by-products. Despite diverse proposed mechanisms and the reported role of facilitator of taxane oxidation (FoTO1) in promoting T-5α-ol formation, conclusive evidence elucidating the catalytic promiscuity of CYP725A4 remains elusive. Here, we report a substrate mimic strategy using taxadien-2-one (taxadienone), which is selectively epoxidized by CYP725A4 at the C4=C5 double bond, suggesting epoxidation as its major catalytic pathway. This notion was then confirmed by the successful identification of the long-observed yet structurally uncharacterized major taxadiene oxidation product as 4(5)α-epoxy-taxadiene, which can spontaneously rearrange in solution. Furthermore, the minor T-5α-ol produced by CYP725A4 arises from a concurrent hydroxylation pathway rather than from epoxidation, and efficient isomerization of 4(5)α-epoxy-taxadiene to T-5α-ol by FoTO1 is required to achieve improved product selectivity. Employing the promiscuity-free taxadienone as a probe substrate eliminated analytical complications and led to variant M3, which shows markedly enhanced catalytic activity through optimized heme-binding and enhanced FG loop flexibility. Finally, co-expressing the plant-derived FoTO1 with the CYP725A4 variant M3 in engineered Escherichia coli increased T-5α-ol production to 109.1 mg/L in a 5 L bioreactor. Collectively, this strategy provides crucial insights into CYP725A4 catalytic promiscuity and enables more efficient directed evolution, thereby facilitating downstream paclitaxel pathway optimization.
- 所属专题：
- 173
