Galectin-8 is a tandem-repeat-type b-galactoside-specific animal lectin possessing N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively), with a difference in carbohydratebinding specificity, involved in cell-matrix interaction, malignant transformation, and cell adhesion. N-CRD shows strong affinity for a2-3-sialylated oligosaccharides, a feature unique to galectin-8. C-CRD usually shows lower affinity for oligosaccharides but higher affinity for N-glycan-type branched oligosaccharides than does N-CRD. There have been many structural studies on galectins with a single carbohydrate recognition domain (CRD), but no X-ray structure of a galectin containing both CRDs has been reported. Here, the X-ray structure of a protease-resistant mutant form of human galectin-8 possessing both CRDs and the novel pseudodimer structure of galectin-8 N-CRD in complexes with a2-3-sialylated oligosaccharide ligands were determined. The results revealed a difference in specificity between N-CRD and C-CRD, and provided new insights into the association of CRDs and/or molecules of galectin-8. DatabaseThe atomic coordinates and structure factors of G8Null-Lac, G8Null-2Lac, G8Null-SiaLacLac, G8N-free and G8N-SiaLacNAc have been deposited in the Protein Data Bank under the accession codes 4FQZ, 3VKL, 3VKM, 3VKN, and 3VKO, respectively. Structured digital abstractG8 and G8 bind by x-ray crystallography (View interaction)

Galectin-9, a tandem-repeat-type ␤-galactoside-specific animal lectin with two carbohydrate recognition domains (CRDs) at the N-and C-terminal ends, is involved in chemoattraction, apoptosis, and the regulation of cell differentiation and has antiallergic effects. Its ability to recognize carbohydrates is essential for its biological functions. Human galectin-9 (hG9) has high affinity for branched N-glycan-type oligosaccharides (dissociation constants of 0.16 -0.70 M) and linear ␤1-3-linked poly-Nacetyllactosamines (0.09 -8.3 M) and significant affinity for the ␣2-3-sialylated oligosaccharides (17-34 M). Further, its N-terminal CRD (hG9N) and C-terminal CRD (hG9C) differ in specificity. To elucidate this unique feature of hG9, x-ray structures of hG9C in the free form and in complexes with N-acetyllactosamine, the biantennary pyridylaminated oligosaccharide, and ␣2-3-sialyllactose were determined. They are the first x-ray structural analysis of C-terminal CRD of the tandem-repeat-type galectin. The results clearly revealed the mechanism by which branched and ␣2-3-sialylated oligosaccharides are recognized and explained the difference in specificity between hG9N and hG9C. Based on structural comparisons with other galectins, we propose that the wide entrance for ligand binding and the shallow binding site of hG9C are favorable for branched oligosaccharides and that Arg 221 is responsible for recognizing sialylated oligosaccharides.The galectins are a family of ␤-galactoside-specific animal lectins that contain conserved elements for carbohydrate recognition (1, 2) and have attracted much attention as novel regulators of the immune system (3). Recently, galectins have been shown to bind glycans on the surface of potentially pathogenic microorganisms and function as recognition and effect factors in innate immunity (4, 5). Currently, there are 14 members of the mammalian galectin family, classified into three subtypes on the basis of structure (6, 7). The prototype (galectin-1, -2, -5, -7, -10, -13, -14, and -15) has a single carbohydrate recognition domain (CRD) 2 that usually forms a non-covalent homodimer. The chimera type (galectin-3) has a single CRD with an extended N-terminal non-lectin domain. The tandem-repeattype galectins (galectin-4, -6, -8, -9, and -12) have two CRDs, one each in the N-and C-terminal regions, joined by a linker peptide.Galectin-9 was independently identified as a tumor antigen in cells of patients with Hodgkin disease (8), as a new type of galectin from a kidney cDNA library (9), and as a novel eosinophil chemoattractant produced by T-lymphocytes (10). It is involved in various biological functions, including chemoattraction (11, 12), apoptosis (13-15), and the regulation of cell differentiation (16, 17), and has anti-allergic effects (18). The chemoattractant activity of galectin-9 depends on its carbohydrate binding activity and requires both CRDs (12). Zhu et al. (19) reported that galectin-9 is a ligand for T-cell immunoglobulin and mucin-containing-protein 3 (TIM3), which is a...

l-Rhamnose isomerase (l-RhI) catalyzes the reversible isomerization of l-rhamnose to l-rhamnulose. Previously determined X-ray structures of l-RhI showed a hydride-shift mechanism for the isomerization of substrates in a linear form, but the mechanism for opening of the sugar-ring is still unclear. To elucidate this mechanism, we determined X-ray structures of a mutant l-RhI in complex with l-rhamnopyranose and d-allopyranose. Results suggest that a catalytic water molecule, which acts as an acid/base catalyst in the isomerization reaction, is likely to be involved in pyranose-ring opening, and that a newly found substrate sub-binding site in the vicinity of the catalytic site may recognize different anomers of substrates.

L-Ribose, a pentose, is not known to exist in nature. Although organisms typically do not have a metabolic pathway that uses L-ribose as a carbon source, prokaryotes use various sugars as carbon sources for survival. Acinetobacter sp. DL-28 has been shown to express the novel enzyme, L-ribose isomerase (AcL-RbI), which catalyzes reversible isomerization between L-ribose and L-ribulose. AcL-RbI showed the highest activity to L-ribose, followed by D-lyxose with 47% activity, and had no significant amino acid sequence similarity to structure-known proteins, except for weak homology with the D-lyxose isomerases from Escherichia coli O157 : H7 (18%) and Bacillus subtilis strain (19%). Thus, AcL-RbI is expected to have the unique three-dimensional structure to recognize L-ribose as its ideal substrate. The X-ray structures of AcL-RbI in complexes with substrates were determined. AcL-RbI had a cupin-type b-barrel structure, and the catalytic site was found between two large b-sheets with a bound metal ion. The catalytic site structures clearly showed that AcL-RbI adopted a cis-enediol intermediate mechanism for the isomerization reaction using two glutamate residues (Glu113 and Glu204) as acid/base catalysts. In its crystal form, AcL-RbI formed a unique homotetramer with many substrate sub-binding sites, which likely facilitated capture of the substrate. DatabaseThe atomic coordinates and structure factors of AcL-RbI/L-ribose, AcL-RbI/L-ribulose, AcLRbI/ribitol, E204Q/L-ribose and E204Q/L-ribulose have been deposited in the Protein Data Bank under accession codes, 4Q0P, 4Q0Q, 4Q0S, 4Q0U and 4Q0V. Structured digital abstract• AcL-RbI and AcL-RbI bind by x-ray crystallography (View interaction).

Acinetobacter sp. l-ribose isomerase (l-RI) catalyzes a reversible isomerization reaction between l-ribose and l-ribulose. To date, information on l-RI remains limited and its amino-acid sequence shows no similarity to those of any known enzymes. Here, recombinant His-tagged l-RI was successfully overexpressed, purified and crystallized. Crystals of His-tagged l-RI were obtained by the hanging-drop vapour-diffusion method at room temperature as two crystal forms which belonged to the monoclinic space group C2, with unit-cell parameters a = 96.60, b = 105.89, c = 71. 83 Å , = 118.16 , and the orthorhombic space group F222, with unit-cell parameters a = 96.44, b = 106.26, c = 117.83 Å . Diffraction data were collected to 3.1 and 2.2 Å resolution, respectively.