基于底物通道的半理性设计实现PaBVMO 催化效率-底物抑制-稳定性的三重协同

Semi-Rational Design of Substrate Channels forPaBVMO to Achieve Triple Synergy of Catalytic Efficiency-Substrate Inhibition-Stability

摘要

来自Pseudomonas aeruginosa 的 Baeyer-Villiger 单加氧酶 (PaBVMO) 能选择性地在10-氧代硬脂酸上羰基的非常规一侧插入氧原子，生成癸二酸辛酯。为突破癸二酸生物合成中PaBVMO 在高底物浓度下催化效率低与底物抑制显著的双重瓶颈，本文提出酶的底物通道半理性重塑策略。通过酶-底物的结合自由能与底物受力分析，解析出底物通道关键门控性环结构。结果表明，获得的最优突变体PaBVMO_G398A 的催化转化数达到 3.45×10⁴，比母本提升了5.3 倍，比活力提升了 0.7 倍，同时失活半衰期从 1.87 h 延长至 6.14 h，稳定性显著增强。通过高斯加速分子动力学 (GaMD) 模拟，解析了 G398A 突变诱导PaBVMO_G398A 的活性口袋发生重排，进而促发PaBVMO_G398A 对 10-氧代硬脂酸催化的底物抑制程度降低、对辅因子 FAD 的亲和力增强、同时稳定性提高的协同机制。

Abstract

Baeyer-Villiger monooxygenase fromPseudomonas aeruginosa (PaBVMO) selectively inserts an oxygen atom into the unconventional side of the carbonyl group of 10-carbonyl stearic acid to produce octyl sebacate, which can be used for synthesizing material monomers sebacic acid and 8-hydroxyoctanoic acid. To break through the dual bottlenecks of low catalytic efficiency and significant substrate inhibition ofPaBVMO under high substrate concentrations in sebacic acid biosynthesis, this study proposed a semi-rational remodeling strategy for the enzyme's substrate channel. By analyzing enzyme-substrate binding free energy and substrate force, the key gating loop structure of the substrate channel was resolved. Based on this strategy, the optimal mutantPaBVMO_G398A was obtained. It exhibited a catalytic turnover number (TON) of 3.45×10⁴, which was 5.3 times higher than that of the parent, and about 0.7 times higher in specific activity. Meanwhile, the inactivation half-life was prolonged from 1.87 h to 6.14 h, and the stability is significantly enhanced. Gaussian-accelerated molecular dynamics (GaMD) simulations further revealed the synergistic mechanism of G398A mutation inducing the rearrangement of the active pocket ofPaBVMO_G398A. This rearrangement effectively alleviated substrate inhibition by 10-carbonyl stearic acid, enhanced affinity for FAD and improved stability.

关键词

癸二酸辛酯 / 生物催化 / PaBVMO / 底物通道重塑 / GaMD

Key words

octyl sebacate / biocatalysis / PaBVMO / substrate channel remodeling / GaMD

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基金资助

国家重点研发计划(2025ZD0615000)

国家自然科学基金(22478116)

上海市关键技术研发计划(25HC2810400)