Structural and Functional Insights into Intramolecular Fructosyl Transfer by Inulin Fructotransferase

Abstract

Inulin fructotransferase (IFTase), a member of glycoside hydrolase family 91, catalyzes depolymerization of ␤-2,1-fructans inulin by successively removing the terminal difructosaccharide units as cyclic anhydrides via intramolecular fructosyl transfer. The crystal structures of IFTase and its substratebound complex reveal that IFTase is a trimeric enzyme, and each monomer folds into a right-handed parallel ␤-helix. Despite variation in the number and conformation of its ␤-strands, the IFTase ␤-helix has a structure that is largely reminiscent of other ␤-helix structures but is unprecedented in that trimerization is a prerequisite for catalytic activity, and the active site is located at the monomer-monomer interface. Results from crystallographic studies and site-directed mutagenesis provide a structural basis for the exolytic-type activity of IFTase and a functional resemblance to inverting-type glycosyltransferases.Fructans are polysaccharides composed of linear and branched polymers of fructose linked to sucrose through glycosidic bonds of various linkage types. They have been conceived as one of the principal stored forms of energy in 15% of higher plants, as well as in a wide range of bacteria and fungi (1). Several plant fructosyltransferases, each with a distinct substrate and glycosidic bond linkage-type specificity, have been suggested to be involved in the sequential enzymatic steps that produce fructans such as ␤-2,6-linked levan and ␤-2,1-linked inulin. In this process, fructose is first linked to vacuolar sucrose, and then fructosyl units are successively added to the resulting trisaccharide (1, 2). Not only do plant fructans play a major role as storage carbohydrates, but they are also implicated in additional physiological functions in plants, such as drought and cold tolerance (1). By contrast, in bacteria, the multifunctional enzymes levansucrase (3) and inulosucrase (4) catalyze fructan biosynthesis, producing inulin and levan, the predominant bacterial fructans, respectively. Details of the levan biosynthetic mechanism in bacteria were recently revealed by structural studies of levansucrase (3, 5).Fructan-degrading enzymes that function primarily in the mobilization of stored fructans in plants and microbes have also been characterized. Just recently, the plant fructan hydrolases (EC 3.2.1) were found to catalyze the hydrolysis of levan and inulin via an exclusively exolytic mechanism that releases successive terminal fructose units (6). The presence of these fructan exohydrolases, even in non-fructan-containing plants, suggests an additional, defensive role for these enzymes against pathogenic bacteria (2).In bacteria, two distinctly different classes of enzymes perform fructan degradation. One of these classes includes two hydrolases, levanase (EC 3.2.1.65) and inulinase (EC 3.2.1.7), which exhibit both endo-and exotype hydrolytic activities. Classifications based on sequence similarity (7) (CAZy; www. cazy.org/CAZY/index.html) place these plant and microbial hydrolytic enzymes into the GH32...

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