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

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

Automated summary

The study focuses on the sequence diversity of Glycoside Hydrolase Family 65 (GH65) and identified specificity-determining positions through in-silico analysis of correlated mutations. By searching for amino acid motifs that differ from previously characterized enzymes, several new clades with potential new functions were identified. Three new enzymes were expressed in E. coli, with one enzyme showing high specificity in hydrolyzing specific substrates.