Rivas-Fernández José Pablo, Vuillemin Marlene, Pilgaard Bo, Klau Leesa J, Fredslund Folmer, Lund-Hanssen Charlotte, Welner Ditte H, Meyer Anne S, Morth J Preben, Meilleur Flora, Aachmann Finn L, Rovira Carme, Wilkens Casper
Departament de Química Inorgànica i Orgànica (secció de Química Orgànica) & IQTCUB, Universitat de Barcelona, Barcelona, Spain.
Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark.
Nat Commun. 2025 Mar 18;16(1):2670. doi: 10.1038/s41467-025-56754-5.
Alginate lyases (ALs) catalyze the depolymerization of brown macroalgae alginates, widely used naturally occurring polysaccharides. Their molecular reaction mechanism remains elusive due to the lack of catalytically competent Michaelis-Menten-like complex structures. Here, we provide structural snapshots and dissect the mechanism of mannuronan-specific ALs from family 7 polysaccharide lyases (PL7), employing time-resolved NMR, X-ray, neutron crystallography, and QM/MM simulations. We reveal the protonation state of critical active site residues, enabling atomic-level analysis of the reaction coordinate. Our approach reveals an endolytic and asynchronous syn β-elimination reaction, with Tyr serving as both Brønsted base and acid, involving a carbanion-type transition state. This study not only reconciles previous structural and kinetic discrepancies, but also establishes a comprehensive PL reaction mechanism which is most likely applicable across all enzymes of the PL7 family as well as other PL families.
海藻酸盐裂解酶（ALs）催化棕色大型海藻海藻酸盐的解聚反应，海藻酸盐是广泛使用的天然存在的多糖。由于缺乏具有催化活性的类似米氏复合物结构，它们的分子反应机制仍然难以捉摸。在这里，我们提供了结构快照，并利用时间分辨核磁共振、X射线、中子晶体学和量子力学/分子力学模拟，剖析了来自7型多糖裂解酶（PL7）家族的甘露糖醛酸特异性ALs的作用机制。我们揭示了关键活性位点残基的质子化状态，从而能够对反应坐标进行原子水平的分析。我们的方法揭示了一种内切且异步的顺式β-消除反应，其中酪氨酸既作为布朗斯特碱又作为酸，涉及一个碳负离子型过渡态。这项研究不仅调和了先前结构和动力学上的差异，还建立了一个全面的PL反应机制，该机制很可能适用于PL7家族的所有酶以及其他PL家族。
