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-wallinvertase1 (AtCWIN1), a key enzyme in sucrose metabolism in plants, catalyzesthe hydrolysis of sucrose into fructose and glucose. AtCWIN1 belongsto the glycoside hydrolase GH-J clan, where two carboxylate residues(Asp23 and Glu203 in AtCWIN1) are well documented as a nucleophileand an acid/base catalyst. However, details at the atomic level aboutthe role of neighboring residues and enzyme-substrate interactionsduring catalysis are not fully understood. Here, quantum mechanical/molecularmechanical (QM/MM) free-energy simulations were carried out to clarifythe origin of the observed decreased rates in Asp239Ala, Asp239Asn,and Asp239Phe in AtCWIN1 compared to the wild type and delineate therole of Asp239 in catalysis. The glycosylation and deglycosylationsteps were considered in both wild type and mutants. Deglycosylationis predicted to be the rate-determining step in the reaction, witha calculated overall free-energy barrier of 15.9 kcal/mol, consistentwith the experimental barrier (15.3 kcal/mol). During the reaction,the -1 furanosyl ring underwent a conformational change correspondingto E-3 & LRARR; [E-2](?) & LRARR; E-1 according to the nomenclature of saccharide structures alongthe full catalytic reaction. Asp239 was found to stabilize not onlythe transition state but also the fructosyl-enzyme intermediate, whichexplains findings from previous structural and mutagenesis experiments.The 1-OH & BULL;& BULL;& BULL;nucleophile interaction has been found toprovide an important contribution to the transition state stabilization,with a contribution of & SIM;7 kcal/mol, and affected glycosylationmore significantly than deglycosylation. This study provides molecularinsights that improve the current understanding of sucrose bindingand hydrolysis in members of clan GH-J, which may benefit proteinengineering research. Finally, a rationale on the sucrose inhibitorconfiguration in chicory 1-FEH IIa, proposed a long time ago in theliterature, is also provided based on the QM/MM calculations.