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

TITLE=Conformational Itinerary of Sucrose During Hydrolysis by Retaining Amylosucrase

JOURNAL=Frontiers in Chemistry

VOLUME=7

YEAR=2019

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2019.00269

DOI=10.3389/fchem.2019.00269

ISSN=2296-2646

ABSTRACT=<p>By means of QM(DFT)/MM metadynamics we have unraveled the hydrolytic reaction mechanism of <italic>Neisseria polysaccharea</italic> amylosucrase (<italic>Np</italic>AS), 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 <sup>4</sup><italic>C</italic><sub>1</sub> conformation, as inferred from structural studies, or a distorted <sup>1</sup><italic>S</italic><sub>3</sub>-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 <sup>4</sup><italic>C</italic><sub>1</sub> 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 (<sup>4</sup><italic>C</italic><sub>1</sub> ad <italic>E</italic><sub>3</sub>) can be viewed as two different states of the glycosyl-enzyme intermediate (GEI), but only the <italic>E</italic><sub>3</sub> state is preactivated for catalysis. These results are consistent with the general conformational itinerary observed for α-glucosidases.</p>