Abstract
Inulin fructotransferase (IFTase), a member of glycoside hydrolase family 91, catalyzes depolymerization of beta-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 substrate-bound complex reveal that IFTase is a trimeric enzyme, and each monomer folds into a right-handed parallel beta-helix. Despite variation in the number and conformation of its beta-strands, the IFTase beta-helix has a structure that is largely reminiscent of other beta-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.
Highlights
Multifunctional enzymes levansucrase [3] and inulosucrase [4] catalyze fructan biosynthesis, producing inulin and levan, the predominant bacterial fructans, respectively
The linkage types of enzymatically produced DFAs vary with the specific enzymes and substrate fructans employed; Inulin fructotransferase (IFTase) produces DFA-I (␣-D-fructofuranose--Dfructofuranose 2Ј,1:2,1Ј-dianhydride) and DFA-III (␣-D-fructofuranose--D-fructofuranose 2Ј,1:2,3Ј-dianhydride), whereas levan fructotransferase results in DFA-IV
The presence of a homotrimeric BsIFTase in a crystalline state is consistent with results obtained from analytical ultracentrifugation experiments (Fig. 2), which indicate that the enzyme exists exclusively as a trimer in solution at concentrations as low as 0.08 mg/ml
Summary
Levan fructotransferases are categorized as GH32 enzymes, like other fructan hydrolases, and they share considerable sequence identity of 30 – 44% and several conserved regions with the other members of GH32 [12, 13]. IFTases do not exhibit any apparent sequence homology with levan fructotransferases, both enzymes carry out similar reactions. IFTases appear to be unrelated to any other known proteins but are highly similar to members of the family, with 50 –98% sequence identity (Fig. 1B), suggesting that the IFTases share a threedimensional structure common to the family. Biochemical features of BsIFTase have been characterized [14, 17], the fundamental details of BsIFTase catalysis, such as the identities of the residues that catalyze intramolecular fructosyl transfer and the mode of enzyme action, remain elusive. Together with the results of site-directed mutagenesis, our structural and functional analyses provide the first atomic resolution structures of IFTase and some details of its enzymatic mechanism
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