Multiscale QM/MM Simulations Identify the Roles of Asp239 and 1‑OH···Nucleophile in Transition State Stabilization in Arabidopsis thaliana Cell-Wall Invertase 1

Arabidopsis thaliana 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 3E ↔ [E2]⧧ ↔ 1E 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.

拟南芥细胞壁蔗糖转化酶1（Arabidopsis thaliana cell-wall invertase 1, AtCWIN1）是植物蔗糖代谢中的关键酶，可催化蔗糖水解为果糖与葡萄糖。AtCWIN1隶属于GH-J族糖苷水解酶（glycoside hydrolase GH-J clan），该族中两个羧基氨基酸残基（AtCWIN1中的Asp23与Glu203）已被广泛证实分别充当亲核试剂与酸碱催化剂。然而，催化过程中邻近残基的作用以及酶-底物相互作用的原子级细节尚未完全明晰。本研究通过量子力学/分子力学（quantum mechanical/molecular mechanical, QM/MM）自由能模拟，阐明了AtCWIN1中Asp239Ala、Asp239Asn与Asp239Phe突变体相较于野生型酶活性降低的分子机制，并厘清了Asp239在催化过程中的作用。研究同时考察了野生型与各突变体的糖基化与去糖基化反应步骤。计算结果表明，去糖基化是该反应的决速步，总自由能垒为15.9 kcal/mol，与实验测得的15.3 kcal/mol自由能垒相符。在完整催化反应过程中，-1位呋喃糖环按照糖类结构命名规则发生了3E ↔ [E2]⧧ ↔ 1E的构象变化，其中[E2]⧧代表过渡态结构。研究发现Asp239不仅可稳定过渡态，还能稳定果糖基-酶中间体，这一结果解释了此前结构生物学与诱变实验的相关发现。1-OH···亲核试剂相互作用可显著稳定过渡态，稳定能约为7 kcal/mol，且该相互作用对糖基化步骤的影响强于去糖基化步骤。本研究从分子层面阐明了GH-J族糖苷水解酶结合蔗糖并催化其水解的机制，加深了学界对该类酶的认知，可为蛋白质工程研究提供参考。最后，本研究基于QM/MM计算，为文献中早前提出的菊苣1-FEH IIa的蔗糖抑制剂构型提供了理论依据。