QM/MM modeling of the hydrolysis and transfructosylation reactions of fructosyltransferase from Aspergillus japonicas, an enzyme that produces prebiotic fructooligosaccharide

Fructosyltransferases (FTs) act on sucrose by cleaving the beta-(2 -> 1) linkage, releasing glucose, and then transferring the fructosyl group to an acceptor molecule. These enzymes are capable of producing prebiotic fructooligosaccharides (FOSs) that are of industrial interest. While several FOS-synthesizing enzymes FTs have been investigated, their catalytic mechanism is not yet fully understood, especially the molecular details of how FOS are enzymatically synthesized from sucrose. Here, we present a comparative quantum mechanics/molecular mechanics (QM/MM) study on the hydrolysis and transfructosylation reactions catalyzed by A. japonicas FT using sucrose as donor and acceptor substrates. It is shown that the hydrolysis and transfructosylation reactions of the enzyme seem to be competitive with similar potential energy profiles. For all studied reaction steps, the fructosyl ring bound in the -1 position was observed to have a E-4 conformation in the oxocarbonium ion-like transition state. Based on the SCC-DFTB/MM simulations of sucrose complexes of wildtype and D191A mutant FT, Asp191 is shown to be responsible for the productive sugar conformation (at subsite -1) required for catalysis. A key interaction, Asp119 center dot center dot center dot nucleophile center dot center dot center dot 1-OH (substrate), is proposed to facilitate the formation of fructosyl-enzyme intermediate. This is the first computational study for understanding the FOS synthesis process, and it can be applicable to related FOS-synthesizing enzymes. (C) 2017 Elsevier Inc. All rights reserved.