Crystal Structure of a Natural Circularly Permuted Jellyroll Protein: 1,3-1,4-β-d-Glucanase from Fibrobacter succinogenes

The 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes (Fsβ-glucanase) is classified as one of the family 16 glycosyl hydrolases. It hydrolyzes the glycosidic bond in the mixed-linked glucans containing β-1,3- and β-1,4-glycosidic linkages. We constructed a truncated form of recombinant Fsβ-glucanase containing the catalytic domain from amino acid residues 1–258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme. The crystal structure of the truncated Fsβ-glucanase was solved at a resolution of 1.7 Å by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein. The overall topology of the truncated Fsβ-glucanase consists mainly of two eight-stranded anti-parallel β-sheets arranged in a jellyroll β-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules. Sequence comparison with other bacterial glucanases showed that Fsβ-glucanase is the only naturally occurring circularly permuted β-glucanase with reversed sequences. Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share ∼70% sequence identity, than to the naturally occurring Fsβ-glucanase of similar topology with 30% identity. This result suggests that protein structure relies more on sequence identity than topology. The high-resolution structure of Fsβ-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability.