Shigehiro Kamitori

Crystal structures and structural comparison of Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) at 1.6 A resolution and alpha-amylase 2 (TVAII) at 2.3 A resolution.

The X-ray crystal structures of Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) and alpha-amylase 2 (TVAII) have been determined at 1.6 A and 2.3 A resolution, respectively. The structures of TVAI and TVAII have been refined, R-factor of 0.182 (R(free)=0.206) and 0.179 (0.224), respectively, with good chemical geometries. Both TVAI and TVAII have four domains, N, A, B and C, and all very similar in structure. However, there are some differences in the structures between them. Domain N of TVAI interacts strongly with domains A and B, giving a spherical shape structure to the enzyme, while domain N of TVAII is isolated from the other domains, which leads to the formation of a dimer. TVAI has three bound Ca ions, whereas TVAII has only one. TVAI has eight extra loops compared to TVAII, while TVAII has two extra loops compared to TVAI. TVAI can hydrolyze substrates more efficiently than TVAII with a high molecular mass such as starch, while TVAII is much more active against cyclodextrins than TVAI and other alpha-amylases. A structural comparison of the active sites has clearly revealed this difference in substrate specificity.

Functional and structural bases of a cysteine-less mutant as a long-lasting substitute for galectin-1.

Galectin-1 (Gal-1), a member of the beta-galactoside-binding animal lectin family, has a wide range of biological activities, which makes it an attractive target for medical applications. Unlike other galectins, Gal-1 is susceptible to oxidation at cysteine residues, which is troublesome for in vitro/vivo studies. To overcome this problem, we prepared a cysteine-less mutant of Gal-1 (CSGal-1) by substituting all cysteine residues with serine residues. In the case of wild-type Gal-1, the formation of covalent dimers/oligomers was evident after 10 days of storage in the absence of a reducing agent with a concomitant decrease in hemagglutination activity, while CSGal-1 did not form multimers and retained full hemagglutination activity after 400 days of storage. Frontal affinity chromatography showed that the sugar-binding specificity and affinity of Gal-1 for model glycans were barely affected by the mutagenesis. Gal-1 is known to induce cell signaling leading to an increase in the intracytoplasmic calcium concentration and to cell death. CSGal-1 is also capable of inducing calcium flux and growth inhibition in Jurkat cells, which are comparable to or more potent than those induced by Gal-1. The X-ray structure of the CSGal-1/lactose complex has been determined. The structure of CSGal-1 is almost identical to that of wild-type human Gal-1, showing that the amino acid substitutions do not affect the overall structure or carbohydrate-binding site structure of the protein. These results indicate that CSGal-1 can serve as a stable substitute for Gal-1.

Complexes of Thermoactinomyces vulgaris R-47 alpha-amylase 1 and pullulan model oligossacharides provide new insight into the mechanism for recognizing substrates with alpha-(1,6) glycosidic linkages.

Thermoactinomyces vulgaris R‐47 α‐amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of α‐(1,4), α‐(1,4), and α‐(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes α‐(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes α‐(1,6) glycosidic linkages with a lesser efficiency. X‐ray structures of three complexes comprising an inactive mutant TVAI (D356N or D356N/E396Q) and a pullulan model oligosaccharide (P2; [Glc‐α‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(1,4)]2 or P5; [Glc‐α‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(1,4)]5) were determined. The complex D356N/P2 is a mimic of the enzyme/product complex in the main catalytic reaction of TVAI, and a structural comparison with Aspergillus oryzaeα‐amylase showed that the (–) subsites of TVAI are responsible for recognizing both starch and pullulan. D356N/E396Q/P2 and D356N/E396Q/P5 provided models of the enzyme/substrate complex recognizing the α‐(1,6) glycosidic linkage at the hydrolyzing site. They showed that only subsites −1 and −2 at the nonreducing end of TVAI are effective in the hydrolysis of α‐(1,6) glycosidic linkages, leading to weak interactions between substrates and the enzyme. Domain N of TVAI is a starch‐binding domain acting as an anchor in the catalytic reaction of the enzyme. In this study, additional substrates were also found to bind to domain N, suggesting that domain N also functions as a pullulan‐binding domain.

X-ray Structures of Human Galectin-9 C-terminal Domain in Complexes with a Biantennary Oligosaccharide and Sialyllactose

Galectin-9, a tandem-repeat-type β-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 anti-allergic 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 μm) and linear β1–3-linked poly-N-acetyllactosamines (0.09–8.3 μm) and significant affinity for the α2–3-sialylated oligosaccharides (17–34 μm). 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 α2–3-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 α2–3-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 Arg221 is responsible for recognizing sialylated oligosaccharides.

Structural analysis of fungus-derived FAD glucose dehydrogenase

We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management.

X-ray structure of a protease-resistant mutant form of human galectin-8 with two carbohydrate recognition domains

Galectin‐8 is a tandem‐repeat‐type β‐galactoside‐specific animal lectin possessing N‐terminal and C‐terminal carbohydrate recognition domains (N‐CRD and C‐CRD, respectively), with a difference in carbohydrate‐binding specificity, involved in cell–matrix interaction, malignant transformation, and cell adhesion. N‐CRD shows strong affinity for α2–3‐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 α2–3‐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.

Carbohydrate recognition mechanism of HA70 from Clostridium botulinum deduced from X-ray structures in complexes with sialylated oligosaccharides

Clostridium botulinum produces the botulinum neurotoxin, forming a large complex as progenitor toxins in association with non‐toxic non‐hemagglutinin and/or several different hemagglutinin (HA) subcomponents, HA33, HA17 and HA70, which bind to carbohydrate of glycoproteins from epithelial cells in the infection process. To elucidate the carbohydrate recognition mechanism of HA70, X‐ray structures of HA70 from type C toxin (HA70/C) in complexes with sialylated oligosaccharides were determined, and a binding assay by the glycoconjugate microarray was performed. These results suggested that HA70/C can recognize both α2–3‐ and α2–6‐sialylated oligosaccharides, and that it has a higher affinity for α2–3‐sialylated oligosaccharides.

Crystal and molecular structure of the γ-cyclodextrin–12-crown-4 1 : 1 inclusion complex

The crystal structure of the γ-cyclodextrin–12-crown-4 1 : 1 inclusion complex has been determined by an X-ray analysis which shows that three γ-cyclodextrins are stacked along a four-fold rotation axis forming a channel-type structure and each γ-cyclodextrin includes a 12-crown-4 molecule in a similar orientation.

Structures of Thermoactinomyces vulgaris R-47 α-Amylase II Complexed with Substrate Analogues

The structures of Thermoactinomyces vulgaris R-47 α-amylase II mutant (d325nTVA II) complexed with substrate analogues, methyl β-cyclodextrin (mβ-CD) and maltohexaose (G6), were solved by X-ray diffraction at 3.2Å and 3.3Å resolution, respectively. In d325nTVA II-mβ-CD complex, the orientation and binding-position of β-CD in TVA II were identical to those in cyclodextin glucanotransferase (CGTase). The active site residues were essentialy conserved, while there are no residues corresponding to Tyr89, Phe183, and His233 of CGTase in TVA II. In d325nTVA II-G6 complex, the electron density maps of two glucosyl units at the non-reducing end were disordered and invisible. The four glucosyl units of G6 were bound to TVA II as in CGTase, while the others were not stacked and were probably flexible. The residues of TVA II corresponding to Tyr89, Lys232, and His233 of CGTase were completely lacking. These results suggest that the lack of the residues related to α-glucan and CD-stacking causes the functional distinctions between CGTase and TVA II.

Crystal structures of open and closed forms of cyclo/maltodextrin-binding protein

The crystal structures of Thermoactinomyces vulgaris cyclo/maltodextrin‐binding protein (TvuCMBP) complexed with α‐cyclodextrin (α‐CD), β‐cyclodextrin (β‐CD) and maltotetraose (G4) have been determined. A common functional conformational change among all solute‐binding proteins involves switching from an open form to a closed form, which facilitates transporter binding. Escherichia coli maltodextrin‐binding protein (EcoMBP), which is structurally homologous to TvuCMBP, has been determined to adopt the open form when complexed with β‐CD and the closed form when bound to G4. Here, we show that, unlike EcoMBP, TvuCMBP–α‐CD and TvuCMBP–β‐CD adopt the closed form when complexed, whereas TvuCMBP–G4 adopts the open form. Only two glucose residues are evident in the TvuCMBP–G4 structure, and these bind to the C‐domain of TvuCMBP in a manner similar to the way in which maltose binds to the C‐domain of EcoMBP. The superposition of TvuCMBP–α‐CD, TvuCMBP–β‐CD and TvuCMBP–γ‐CD shows that the positions and the orientations of three glucose residues in the cyclodextrin molecules overlay remarkably well. In addition, most of the amino acid residues interacting with these three glucose residues also participate in interactions with the two glucose residues in TvuCMBP–G4, regardless of whether the protein is in the closed or open form. Our results suggest that the mechanisms by which TvuCMBP changes from the open to the closed conformation and maintains the closed form appear to be different from those of EcoMBP, despite the fact that the amino acid residues responsible for the initial binding of the ligands are well conserved between TvuCMBP and EcoMBP.