Wang Jiawei, Ouyang Xingyu, Meng Shiyu, Li Jiayi, Liu Liangxu, Li Chaofeng, Li Hengrun, Zheng Haotian, Liao Chao, Zhao Yi-Lei, Ni Jun
State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, China.
iScience. 2024 Dec 10;28(1):111570. doi: 10.1016/j.isci.2024.111570. eCollection 2025 Jan 17.
Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of Ado, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes.
木质素的高值化对于实现经济且可持续的生物精炼过程至关重要。然而，参与木质素降解的酶对底物的偏好仍知之甚少，且对特定底物的低活性给木质素的有效利用带来了重大挑战。在本研究中，我们调查了参与木质素高值化的关键酶Ado的底物混杂性。反应前状态分析表明，氢键网络在决定底物选择性方面至关重要。通过对底物通道周围的残基进行靶向饱和诱变，我们鉴定出Y205W和Y205Q突变体，其活性分别提高了0.73倍和0.72倍。结构分析表明，原始底物通道的重新定向可能是活性提高的原因。我们的研究为木质素降解酶的底物偏好机制提供了重要见解，并表明这种通道重定向策略可扩展到其他混杂性酶。
