Crystal Structure of Exo-inulinase from Aspergillus awamori: The Enzyme Fold and Structural Determinants of Substrate Recognition

Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked β-d-fructofuranose residues in inulin, levan and sucrose releasing β-d-fructose. We present the X-ray structure at 1.55 Å resolution of exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase family 32, solved by single isomorphous replacement with the anomalous scattering method using the heavy-atom sites derived from a quick cryo-soaking technique. The tertiary structure of this enzyme folds into two domains: the N-terminal catalytic domain of an unusual five-bladed β-propeller fold and the C-terminal domain folded into a β-sandwich-like structure. Its structural architecture is very similar to that of another member of glycoside hydrolase family 32, invertase (β-fructosidase) from Thermotoga maritima, determined recently by X-ray crystallography The exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87 Å, reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition.