First crystal structure of an endo-levanase - the BT1760 from a human gut commensal Bacteroides thetaiotaomicron.

ABSTRACT: The endo-levanase BT1760 of a human gut commensal Bacteroides thetaiotaomicron randomly cuts a ?-2,6-linked fructan, levan, into fructo-oligosaccharides providing a prebiotic substrate for gut microbiota. Here we introduce the crystal structure of BT1760 at resolution of 1.65?Å. The fold of the enzyme is typical for GH32 family proteins: a catalytic N-terminal five-bladed ?-propeller connected with a C-terminal ?-sandwich domain. The levantetraose-bound structure of catalytically inactive mutant E221A at 1.90-Å resolution reveals differences in substrate binding between the endo-acting fructanases. A shallow substrate-binding pocket of the endo-inulinase INU2 of Aspergillus ficuum binds at least three fructose residues at its flat bottom. In the levantetraose-soaked crystal of the endo-levanase E221A mutant the ligand was bent into the pond-like substrate pocket with its fructose residues making contacts at -3, -2, -1 and?+?1 subsites residing at several pocket depths. Binding of levantetraose to the ?-sandwich domain was not detected. The N- and C-terminal modules of BT1760 did not bind levan if expressed separately, the catalytic domain lost its activity and both modules tended to precipitate. We gather that endo-levanase BT1760 requires both domains for correct folding, solubility and stability of the protein.

SUBMITTER: Ernits K 

PROVIDER: S-EPMC6560043 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature