Conformational Itinerary of Sucrose During Hydrolysis by Retaining Amylosucrase.

Santiago Alonso-Gil,Carme Rovira,Isabelle André,Joan Coines

doi:10.3389/fchem.2019.00269

Abstract

By means of QM(DFT)/MM metadynamics we have unraveled the hydrolytic reaction mechanism of Neisseria polysaccharea amylosucrase (NpAS), a member of GH13 family. Our results provide an atomistic picture of the active site reorganization along the catalytic double-displacement reaction, clarifying whether the glycosyl-enzyme reaction intermediate features an α-glucosyl unit in an undistorted 4C1 conformation, as inferred from structural studies, or a distorted 1S3-like conformation, as expected from mechanistic analysis of glycoside hydrolases (GHs). We show that, even though the first step of the reaction (glycosylation) results in a 4C1 conformation, the α-glucosyl unit undergoes an easy conformational change toward a distorted conformation as the active site preorganizes for the forthcoming reaction step (deglycosylation), in which an acceptor molecule, i.e., a water molecule for the hydrolytic reaction, performs a nucleophilic attack on the anomeric carbon. The two conformations (4C1 ad E3) can be viewed as two different states of the glycosyl-enzyme intermediate (GEI), but only the E3 state is preactivated for catalysis. These results are consistent with the general conformational itinerary observed for α-glucosidases.

Highlights

Glycoside hydrolases (GHs) are enzymes responsible for the degradation or hydrolysis of glycosidic bonds in carbohydrates
Our results provide an atomistic picture of the active site reorganization along the catalytic double-displacement reaction, clarifying whether the glycosyl-enzyme reaction intermediate features an α-glucosyl unit in an undistorted 4C1 conformation, as inferred from structural studies, or a distorted 1S3-like conformation, as expected from mechanistic analysis of glycoside hydrolases (GHs)
Even though the first step of the reaction results in a 4C1 conformation, the α-glucosyl unit undergoes an easy conformational change toward a distorted conformation as the active site preorganizes for the forthcoming reaction step, in which an acceptor molecule, i.e., a water molecule for the hydrolytic reaction, performs a nucleophilic attack on the anomeric carbon

Summary

Introduction

Glycoside hydrolases (GHs) are enzymes responsible for the degradation or hydrolysis of glycosidic bonds in carbohydrates. The substrate conformational itinerary from the Michaelis complex (MC, Figure S1) to the covalent glycosyl-enzyme intermediate (GEI), is expected to be identical.

Results

Conclusion