Ice-binding proteins (IBPs) inhibit ice growth through direct interaction with ice crystals to permit the survival of polar organisms in extremely cold environments. FfIBP is an ice-binding protein encoded by the Antarctic bacterium Flavobacterium frigoris PS1. The X-ray crystal structure of FfIBP was determined to 2.1 Å resolution to gain insight into its ice-binding mechanism. The refined structure of FfIBP shows an intramolecular disulfide bond, and analytical ultracentrifugation and analytical size-exclusion chromatography show that it behaves as a monomer in solution. Sequence alignments and structural comparisons of IBPs allowed two groups of IBPs to be defined, depending on sequence differences between the α2 and α4 loop regions and the presence of the disulfide bond. Although FfIBP closely resembles Leucosporidium (recently re-classified as Glaciozyma) IBP (LeIBP) in its amino-acid sequence, the thermal hysteresis (TH) activity of FfIBP appears to be tenfold higher than that of LeIBP. A comparison of the FfIBP and LeIBP structures reveals that FfIBP has different ice-binding residues as well as a greater surface area in the ice-binding site. Notably, the ice-binding site of FfIBP is composed of a T-A/G-X-T/N motif, which is similar to the ice-binding residues of hyperactive antifreeze proteins. Thus, it is proposed that the difference in TH activity between FfIBP and LeIBP may arise from the amino-acid composition of the ice-binding site, which correlates with differences in affinity and surface complementarity to the ice crystal. In conclusion, this study provides a molecular basis for understanding the antifreeze mechanism of FfIBP and provides new insights into the reasons for the higher TH activity of FfIBP compared with LeIBP.

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...