Yang Liu, Pu Zhongji, Wu Jianping, Liu Xiaofeng, Wang Zhe, Yu Haoran, Wang Liuwei, Meng Yan, Xu Gang, Yang Lirong, Zheng Wenlong
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310058, China.
ZJU-Hangzhou Global Scientific and Technological Innovation Centre, Zhejiang University, Hangzhou, 311215, China.
Nat Commun. 2025 Jul 14;16(1):6494. doi: 10.1038/s41467-025-61773-3.
N-oxidation of N-heterocycles is essential in the synthesis of natural products but challenging due to low efficacy and poor regioselectivity. In this study, the N-oxidation selective potential of P450BM3 from Bacillus megaterium for N-heterocyclic compounds is investigated. Here, twelve amino acids located in the active center, including A74, L75, V78, A82, F87, I263, A264, A328, P329, A330, I401, and L437, are investigated by site-saturation mutation. As a result, F87, A264, L75, V78, A328, I401, and L437 are identified as hotspot residues. Subsequently, the combinatorial active-site saturation test/iterative saturation mutagenesis strategy is performed. Using quinoline as a model substrate, the mutant F87G/A264G/A328L exhibits N-oxidation selectivity of up to 99.0%, with a conversion rate of 99.3%. Molecular dynamics simulations uncover a "push-pull" molecular mechanism elucidating the pivotal role of steric factors in determining substrate recognition and N-oxidation selectivity. This study provides an efficient N-oxide synthesis method and insights into P450BM3's molecular mechanisms.
N-杂环的N-氧化在天然产物合成中至关重要，但由于效率低和区域选择性差而具有挑战性。在本研究中，研究了巨大芽孢杆菌的P450BM3对N-杂环化合物的N-氧化选择性潜力。在此，通过位点饱和突变研究了位于活性中心的12个氨基酸，包括A74、L75、V78、A82、F87、I263、A264、A328、P329、A330、I401和L437。结果，F87、A264、L75、V78、A328、I401和L437被确定为热点残基。随后，进行了组合活性位点饱和测试/迭代饱和诱变策略。以喹啉为模型底物，突变体F87G/A264G/A328L表现出高达99.0%的N-氧化选择性，转化率为99.3%。分子动力学模拟揭示了一种“推-拉”分子机制，阐明了空间因素在决定底物识别和N-氧化选择性中的关键作用。本研究提供了一种高效的N-氧化物合成方法，并深入了解了P450BM3的分子机制。
