Hydrolysis of Glycosyl Thioimidates by Glycoside Hydrolase Requires Remote Activation for Efficient Activity

Guillotin Guillotin,Daniellou Daniellou,Assaf Assaf,Pistorio Pistorio,Lafite Lafite,Demchenko Demchenko

doi:10.3390/catal9100826

Abstract

Chemoenzymatic synthesis of glycosides relies on efficient glycosyl donor substrates able to react rapidly and efficiently, yet with increased stability towards chemical or enzymatic hydrolysis. In this context, glycosyl thioimidates have previously been used as efficient donors, in the case of hydrolysis or thioglycoligation. In both cases, the release of the thioimidoyl aglycone was remotely activated through a protonation driven by a carboxylic residue in the active site of the corresponding enzymes. A recombinant glucosidase (DtGly) from Dictyoglomus themophilum, previously used in biocatalysis, was also able to use such glycosyl thioimidates as substrates. Yet, enzymatic kinetic values analysis, coupled to mutagenesis and in silico modelling of DtGly/substrate complexes demonstrated that the release of the thioimidoyl moiety during catalysis is only driven by its leaving group ability, without the activation of a remote protonation. In the search of efficient glycosyl donors, glycosyl thioimidates are attractive and efficient. Their utility, however, is limited to enzymes able to promote leaving group release by remote activation.

Highlights

Enzymes proved to be efficient synthetic tools for the eco-compatible synthesis of many classes of compounds
We demonstrated that DtGly, a glycoside hydrolases (GH) previously used in chemoenzymatic synthesis of
Our previous study demonstrated that DtGly could be used in chemoenzymatic synthesis of glycosides, thereby serving as an attractive biocatalyst that needed to be assayed for other substrates [36]

Summary

Introduction

Enzymes proved to be efficient synthetic tools for the eco-compatible synthesis of many classes of compounds. Genetic modifications of recombinant enzymes are powerful tools to alter versatility and properties of the engineered proteins. A vast array of carbohydrate-metabolizing enzymes (CAZYmes), including glycoside hydrolases (GH) or glycosyltransferases (GT), has been engineered and used for the chemo-enzymatic synthesis of glycosides [3]. The corresponding methodologies have proved useful in numerous applications ranging from glycosylated natural products to pharmaceuticals [4,5]. Only few examples in the literature have been describing the use of CAZYmes for the preparation of synthetic thioglycosides that exhibit a sulphur atom linking the glycone and aglycone counterparts instead of more conventional oxygen or nitrogen atoms [6]. When compared to the corresponding O-glycosides, S-glycosides are highly stable towards enzymatic and acidic hydrolyses

Methods

Results

Discussion

Conclusion