Publications

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

  De Beul E., Jongbloet A., Franceus J., Desmet T.

  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 w ... >> More

  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 <i>E. coli</i> and their substrate profile was mapped. One of those enzymes, originating from the bacterium <i>Mucilaginibacter mallensis</i>, 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. << Less

  Molecules 26:0-0(2021) [PubMed] [EuropePMC]

• Structure of a bacterial alpha-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases.

  Nakamura S., Nihira T., Kurata R., Nakai H., Funane K., Park E.Y., Miyazaki T.

  Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no cr ... >> More

  Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu⁴⁷² was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu⁶¹⁶ instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family. << Less

  J. Biol. Chem. 297:101366-101366(2021) [PubMed] [EuropePMC]