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 β-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 β-propeller connected with a C-terminal β-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 β-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.

Insights into SusCD-mediated glycan import by a prominent gut symbiont

“…2a ). The bound oligosaccharide is compact and has a twisted, somewhat helical conformation, similar to that observed for levantetraose in complex with the endo-levanase 26 . The ligand makes numerous polar contacts with side chains of residues in both Bt1762 and Bt1763 (Fig.…”

Diversity and Function of Phage Encoded Depolymerases

“…Again, it seems that also levP was acquired from a host organism. LevP is not a TSP, because similar levanases rather exhibit an N-terminal beta-propeller and a C-terminal beta-sandwich (Ernits et al, 2019). Apparently, PghP-and LevP-like proteins are also encoded in many other bacteriophages infecting B. subtilis (Ozaki et al, 2017).…”

Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans

“…The pURI3-BaAG2Cter and pURI-ScMAL62Cter containing the BaAG2 gene or MAL62 gene, respectively, were electroporated into E. coli BL21 (DE3) for heterologous expression. A simplified autoinduction medium as in [64] was used for protein overproduction: the LB-based medium (20 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) was supplemented with 25 mM phosphate buffer (Na 2 HPO 4 /KH 2 PO 4 ; pH 7.2) and 3 g/L glycerol to which filter-sterilized 0.25 g/L glucose and 1 g/L lactose were added. Medium for transformant selection contained ampicillin (Amp, 150 mg/L) for plasmid preservation.…”