祖先序列重建与理性设计赋能非特异性过加氧酶实现高效甾体母核氧化官能团化

doi:10.1016/S1872-2067(26)64991-1

催化学报

祖先序列重建与理性设计赋能非特异性过加氧酶实现高效甾体母核氧化官能团化

胡瑞文¹, 郭志勇¹, 彭小刚¹, 石晓琪, 乔玉本, 李倩, 高成华, 李爱涛^*

湖北大学生命科学学院, 工业生物技术湖北省重点实验室, 省部共建生物催化与酶工程国家重点实验室, 湖北武汉 430062

收稿日期:2025-09-10 接受日期:2025-11-16
通讯作者: *电子信箱: aitaoli@hubu.edu.cn (李爱涛).
作者简介:¹共同第一作者.
基金资助:
国家重点研发计划(2024YFA0917800); 湖北武汉市自然科学基金(2024040701010046); 湖北省学科创新引智基地(2019BJH021); 湖北省高等学校优秀中青年科技创新团体计划(T2024001).

Synergistic integration of ancestral sequence reconstruction and rational design empowers unspecific peroxygenase for efficient steroid core oxyfunctionalization

Ruiwen Hu¹, Zhiyong Guo¹, Xiaogang Peng¹, Xiaoqi Shi, Yuben Qiao, Qian Li, Chenghua Gao, Aitao Li^*

State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of life science, Hubei University, Wuhan 430062, Hubei, China

Received:2025-09-10 Accepted:2025-11-16
Contact: * E-mail: aitaoli@hubu.edu.cn (A. Li).
About author:¹ Contributed to this work equally.
Supported by:
National Key Research and Development Program of China (2024YFA0917800), the Natural Science Foundation of Wuhan City (2024040701010046), the Innovation Base for Introducing Talents of Discipline of Hubei Province (2019BJH021), and the Outstanding Science and Technology Innovation Team Program for Young and Middle-aged Scholars in Higher Education Institutions of Hubei Province (T2024001).

摘要/Abstract

摘要： 甾体母核羟基化是药物合成中的关键步骤, 可用于制备具有生物活性的皮质类固醇及激素衍生物. 然而, 传统化学方法存在反应条件苛刻、区域选择性差以及环境污染等问题. 尽管以细胞色素P450酶为代表的生物催化途径提供了绿色的替代方案, 但其对昂贵辅因子(NADPH)和复杂电子传递系统的依赖限制了其工业应用. 相比之下, 非特异性过加氧酶(UPOs)仅需H₂O₂作为氧供体与电子受体, 是目前最具发展潜力的氧化酶之一. 然而其对甾体母核羟基化的天然催化效率与选择性差, 无法满足实际应用需求. 目前, CglUPO是少数具有甾体母核羟基化活性的UPO, 但其低活性及环氧化物副产物的生成阻碍了其应用. 因此, 开发具有更高活性和高区域选择性的UPO酶对推进绿色甾体药物合成至关重要.
为突破现有瓶颈, 本研究集成祖先序列重建(ASR)与结构引导的理性设计, 成功创制出高性能UPO变体, 实现了甾体母核的高效选择性羟基化. 我们首先通过ASR技术复活了祖先酶N1, 与目前研究最深入的甾体羟基化UPO(CglUPO)相比, N1的表达量提升了3倍, 并对底物雌甾-4,9-二烯-3,17-二酮展现出中等水平的11β-区域选择性(63%). 其次, 基于分子动力学模拟, 对关键底物结合残基进行理性突变,获得了双突变N1-F83G/L232V. 该突变体在活性(提高超36倍)和区域选择性(C11β-羟化> 99%)方面表现出显著提升. 机理研究表明, L232V突变通过减少空间位阻来促进底物结合; 而F83G则通过消除有害的疏水锚点, 修正了底物在活性中心内的取向. 这一发现挑战了“疏水相互作用必然有益于催化”的传统认知. 最后, 为评估其应用潜力, 我们考察了该变体对不同甾体底物的催化普适性. 结果表明, N1-F83G/L232V能够根据不同底物的结构特征, 精准地合成11β-, 16α-和6β-等多种羟基化产物, 展现出宽广的底物适用范围. 在此基础上, 我们成功完成了克级规模的合成验证. 其中关键药物中间体11β-羟基-雌甾-4,9-二烯-3,17-二酮(合成去氧孕烯的关键前体)的分离收率达到86%; 16α-羟基-诺龙(合成雌三醇的关键前体)收率为80%; 6β-羟基-1,4-二烯-3,17-二酮(合成依西美坦的关键前体)收率为40%. 这一可放大性充分证明了其工业应用的可行性.
综上, 本研究不仅为甾体药物的绿色合成提供了强大且可放大的生物催化工具, 更通过ASR与理性设计的成功融合, 重塑了UPO的酶工程范式. 所揭示的独特催化机理为后续设计提供了全新理论指导. 鉴于其在解决实际合成难题与推动可持续制造方面的巨大潜力, 有望在酶工程、药物化学及绿色生物制造领域引发广泛关注.

关键词: 甾体羟化, 非特异性过加氧酶, 祖先序列重建, 区域/立体选择性

Abstract: Unspecific peroxygenases (UPOs) are versatile biocatalysts for selective oxyfunctionalization, yet their use in steroid core hydroxylation remains underdeveloped. Although CglUPO is the best-studied steroid-hydroxylating UPO, its inefficiency and tendency to form unwanted epoxides limit its practicality. To overcome this, we employed ancestral sequence reconstruction (ASR) to obtain ancestral UPO N1 that exhibited 3-fold higher expression than modern counterparts CglUPO and demonstrated moderate regioselectivity (63%) for 11β-hydroxylation of estra-4,9-diene-3,17-dione (1). Guided by molecular dynamics simulations, rational mutagenesis of key substrate-binding residues generated the variant N1-F83G/L232V. This variant achieved a 36-fold increase in catalytic activity with near-complete 11β-regioselectivity (99%). Mechanistic study revealed that L232V reduces steric hindrance near the active site, while F83G eliminates a mispositioned hydrophobic anchor—challenging the paradigm that hydrophobic interactions always benefit catalysis. Substrate scope studies confimred the broad applicability of this variant, yielding different hydroxylation patterns (11β-, 16α-, 6β-) depending on steroids tested. Gram-scale synthesis afforded isolated yields of 40~86% for different hydroxylated steriods, which serve as pivotal intermediates for synthesizing desogestrel, estriol, and exemestane. Overall, this work advances the steroid biocatalysis toolbox, demonstrates how integraing ASR with rational engineering solves UPO limitations, and establishes UPOs as industrially viable catalysts for steroid pharmaceutical synthesis.

Key words: Steroid hydroxylation, Unspecific peroxygenases, Acestral sequence reconstruction, Regioselectivity

胡瑞文, 郭志勇, 彭小刚, 石晓琪, 乔玉本, 李倩, 高成华, 李爱涛. 祖先序列重建与理性设计赋能非特异性过加氧酶实现高效甾体母核氧化官能团化[J]. 催化学报, DOI: 10.1016/S1872-2067(26)64991-1.

Ruiwen Hu, Zhiyong Guo, Xiaogang Peng, Xiaoqi Shi, Yuben Qiao, Qian Li, Chenghua Gao, Aitao Li. Synergistic integration of ancestral sequence reconstruction and rational design empowers unspecific peroxygenase for efficient steroid core oxyfunctionalization[J]. Chinese Journal of Catalysis, DOI: 10.1016/S1872-2067(26)64991-1.

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