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

Abstract

The Glycoside Hydrolase Family 65 (GH65) is an enzyme family of inverting a-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 a-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.

Identification and Characterization of Dextran α-1,2-Debranching Enzyme from <i>Microbacterium dextranolyticum</i>

“… 43) 44) 45) Kojibiose hydrolase, a member of GH65 that hydrolyzes α-(1→2)-glucosidic linkage in kojioligosaccharides, has recently been discovered in bacteria. 46) 47) A third protein was revealed to belong to GH97, where anomer-inverting α-glucoside hydrolases, anomer-retaining α-galactosidases, and anomer-retaining β-L-arabinopyranosidase have been found. 48) 49) 50) However, the GH97 protein lacks a putative signal peptide in contrast to the other two proteins found and shares a substantially larger degree of sequence identity with retaining α-galactosidases than with inverting α-glucoside hydrolases.…”

“…GH65 consists mainly of GPs from bacteria and GHs from eukaryotes that act on α-glucosidic linkages, 43) 44) 45) and recently the first bacterial GH, kojibiose hydrolase (EC 3.2.1.216), was discovered in Flavobacterium johnsoniae (FjGH65A) and Mucilaginibacter mallensis . 46) 47) Moreover, the biochemical and crystallographic studies of FjGH65A revealed catalytic residues and the anomer-inverting mechanism of GH65 GHs. 46) MdDDE was identified as a GH that released glucose from α-(1→2)-branched α-glucan in the present study and is therefore the second bacterial GH65 GH following kojibiose hydrolase.…”