Discovery of a Kojibiose Hydrolase by Analysis of Specificity-Determining Correlated Positions in Glycoside Hydrolase Family 65.

Emma De Beul,Jorick Franceus,Tom Desmet,Alana Jongbloet

doi:10.3390/molecules26206321

Abstract

The Glycoside Hydrolase Family 65 (GH65) is an enzyme family of inverting α-glucoside phosphorylases and hydrolases that currently contains 10 characterized enzyme specificities. However, its sequence diversity has never been studied in detail. Here, an in-silico analysis of correlated mutations was performed, revealing specificity-determining positions that facilitate annotation of the family’s phylogenetic tree. By searching these positions for amino acid motifs that do not match those found in previously characterized enzymes from GH65, several clades that may harbor new functions could be identified. Three enzymes from across these regions were expressed in E. coli and their substrate profile was mapped. One of those enzymes, originating from the bacterium Mucilaginibacter mallensis, was found to hydrolyze kojibiose and α-1,2-oligoglucans with high specificity. We propose kojibiose glucohydrolase as the systematic name and kojibiose hydrolase or kojibiase as the short name for this new enzyme. This work illustrates a convenient strategy for mapping the natural diversity of enzyme families and smartly mining the ever-growing number of available sequences in the quest for novel specificities.

Highlights

In the carbohydrate-active enzymes database (CAZy) [1], 10 enzyme specificities (Table 1) are grouped to form the Glycoside Hydrolase Family 65 (GH65), which is a member of clan GH-L
GH65 enzymes are typically active on α-glucobioses or derivatives thereof, but a few other natural substrates have been identified (3-O-α-glucosyl-L-rhamnose, 2-O-αglucosylglycerol or the α-glucosyl-1,2-β-galactosyl decoration found on hydroxylysine residues of collagen) [2,4,5,6]
We describe the discovery of a kojibiose glucohydrolase from the bacterium Mucilaginibacter mallensis

Summary

Introduction

In the carbohydrate-active enzymes database (CAZy) [1], 10 enzyme specificities (Table 1) are grouped to form the Glycoside Hydrolase Family 65 (GH65), which is a member of clan GH-L. All members of this enzyme family break α-glucosidic bonds through a single displacement mechanism that inverts the anomeric configuration (Figure 1), but they differ in the substrates they prefer [2]. Based on the nucleophile that is used in the breakdown reaction, two groups can be distinguished: glucoside hydrolases use water to hydrolyze the glucosidic bond, whereas glucoside phosphorylases employ inorganic phosphate, releasing glucose or β-glucose 1-phosphate (βGlc1P) as a reaction product, respectively [3].

Methods

Results

Conclusion