Demonstration School, University of Phayao, Phayao 56000, Thailand.
Unit of Excellence in Computational Molecular Science and Catalysis, and Division of Chemistry, School of Science, University of Phayao, Phayao 56000, Thailand.
J Chem Inf Model. 2023 Aug 14;63(15):4827-4838. doi: 10.1021/acs.jcim.3c00796. Epub 2023 Jul 28.
cell-wall invertase 1 (AtCWIN1), a key enzyme in sucrose metabolism in plants, catalyzes the hydrolysis of sucrose into fructose and glucose. AtCWIN1 belongs to the glycoside hydrolase GH-J clan, where two carboxylate residues (Asp23 and Glu203 in AtCWIN1) are well documented as a nucleophile and an acid/base catalyst. However, details at the atomic level about the role of neighboring residues and enzyme-substrate interactions during catalysis are not fully understood. Here, quantum mechanical/molecular mechanical (QM/MM) free-energy simulations were carried out to clarify the origin of the observed decreased rates in Asp239Ala, Asp239Asn, and Asp239Phe in AtCWIN1 compared to the wild type and delineate the role of Asp239 in catalysis. The glycosylation and deglycosylation steps were considered in both wild type and mutants. Deglycosylation is predicted to be the rate-determining step in the reaction, with a calculated overall free-energy barrier of 15.9 kcal/mol, consistent with the experimental barrier (15.3 kcal/mol). During the reaction, the -1 furanosyl ring underwent a conformational change corresponding to E ↔ [E] ↔ E according to the nomenclature of saccharide structures along the full catalytic reaction. Asp239 was found to stabilize not only the transition state but also the fructosyl-enzyme intermediate, which explains findings from previous structural and mutagenesis experiments. The 1-OH···nucleophile interaction has been found to provide an important contribution to the transition state stabilization, with a contribution of ∼7 kcal/mol, and affected glycosylation more significantly than deglycosylation. This study provides molecular insights that improve the current understanding of sucrose binding and hydrolysis in members of clan GH-J, which may benefit protein engineering research. Finally, a rationale on the sucrose inhibitor configuration in chicory 1-FEH IIa, proposed a long time ago in the literature, is also provided based on the QM/MM calculations.
细胞壁转化酶 1（AtCWIN1）是植物蔗糖代谢中的关键酶，可将蔗糖水解为果糖和葡萄糖。AtCWIN1 属于糖苷水解酶 GH-J 家族，该家族中有两个羧酸根残基（AtCWIN1 中的天冬氨酸 23 和谷氨酸 203）被很好地记录为亲核试剂和酸碱催化剂。然而，关于催化过程中邻近残基和酶-底物相互作用的原子水平细节尚不完全清楚。在此，进行了量子力学/分子力学（QM/MM）自由能模拟，以阐明与野生型相比，AtCWIN1 中的 Asp239Ala、Asp239Asn 和 Asp239Phe 观察到的降低的速率的起源，并阐明 Asp239 在催化中的作用。在野生型和突变体中都考虑了糖基化和去糖基化步骤。预测去糖基化是反应的速率决定步骤，总自由能势垒为 15.9 kcal/mol，与实验势垒（15.3 kcal/mol）一致。在反应过程中，根据糖结构的命名法，-1 呋喃糖环经历了构象变化，对应于 E↔[E]↔E。发现 Asp239 不仅稳定了过渡态，还稳定了果糖-酶中间物，这解释了先前结构和突变实验的结果。1-OH···亲核试剂相互作用已被发现为过渡态稳定提供了重要贡献，贡献约为 7 kcal/mol，并且对糖基化的影响比对去糖基化的影响更大。这项研究提供了分子见解，提高了对 GH-J 家族成员中蔗糖结合和水解的现有理解，这可能有益于蛋白质工程研究。最后，还根据 QM/MM 计算为文献中很久以前提出的菊苣 1-FEH IIa 中蔗糖抑制剂的构象提供了依据。
