Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on beta-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form alpha-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (alpha-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [alpha FFase1]) as an enzyme with alpha-D-fructofuranosidase and alpha-D-arabinofuranosidase activities and an anomer-retaining manner. alpha FFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with alpha FFase1, conversions of beta-D-Frup-(2 -> 1)-alpha-D-Fruf to alpha-D-Fruf-1,2':2,1'-beta-D-Frup (diheterolevulosan II) and beta-D-Fruf-(2 -> 1)-alpha-D-Fruf (inulobiose) to alpha-D-Fruf-1,2':2,1'-beta- D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of alpha FFase1 in complex with beta-D-Fruf and beta-D-Araf were determined at the resolutions of up to 1.76 A. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of alpha FFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).

Automated summary

A novel glycoside hydrolase family member, alpha-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase, was identified in Bifidobacterium dentium. This enzyme has unique activities and is not homologous with known enzymes, indicating its novelty in the field of glycoside hydrolases.