Atsuo Kimura | Hokkaido University (original) (raw)

Papers by Atsuo Kimura

FEBS letters, Jan 25, 2014

The role of calcium ion in the active site of the inverting glycoside hydrolase family 97 enzyme,... more The role of calcium ion in the active site of the inverting glycoside hydrolase family 97 enzyme, BtGH97a, was investigated through structural and kinetic studies. The calcium ion was likely directly involved in the catalytic reaction. The pH dependence of kcat/Km values in the presence or absence of calcium ion indicated that the calcium ion lowered the pKa of the base catalyst. The significant decreases in kcat/Km for hydrolysis of substrates with basic leaving groups in the absence of calcium ion confirmed that the calcium ion facilitated the leaving group departure.

Bioscience, biotechnology, and biochemistry, 2013

Acarbose is a pseudo-tetrasaccharide and one of the most effective inhibitors of glycoside hydrol... more Acarbose is a pseudo-tetrasaccharide and one of the most effective inhibitors of glycoside hydrolases. Its derivatives, acarviosyl-maltooligosaccharides, which have longer maltooligosaccharide parts than the maltose unit of acarbose, were synthesized using a disproportionating enzyme partially purified from adzuki cotyledons. The enzyme was identified as a typical type-1 disproportionating enzyme (DPE1) by primary structure analysis. It produced six compounds from 100 mM acarbose and 7.5% (w/v) of maltotetraose-rich syrup. The masses of the six products were confirmed to accord with acarviosyl-maltooligosaccharides with the degrees of polymerization = 5-10 (AC5-AC10) by electrospray ionization mass spectrometry. (1)H and (13)C NMR spectra indicated that AC5-AC10 were α-acarviosyl-(1→4)-maltooligosaccharide, which have maltotriose-maltooctaose respectively in the maltooligosaccharide part. A predominance of AC7 in the products at the early stage of the reaction indicated that DPE1 ca...

Journal of biochemistry, Jan 21, 2015

The crystal structures of the wild-type and catalytic mutant Asp-312→Gly in complex with isomalto... more The crystal structures of the wild-type and catalytic mutant Asp-312→Gly in complex with isomaltohexaose of endo-1,6-dextranase from the thermophilic bacterium Thermoanaerobacter pseudethanolicus (TpDex), belonging to the glycoside hydrolase family 66, were determined. TpDex consists of three structural domains, a catalytic domain comprising an (β/α)8-barrel and two β-domains located at both N- and C-terminal ends. The isomaltohexaose-complex structure demonstrated that the isomaltohexaose molecule was bound across the catalytic site, showing that TpDex had 6 subsites (-4 to +2) in the catalytic cleft. Marked movement of the Trp-376 side-chain along with loop 6, which was the side wall component of the cleft at subsite +1, was observed to occupy subsite +1, indicating that it might expel the cleaved aglycone subsite after the hydrolysis reaction. Structural comparison with other mesophilic enzymes indicated that several structural features of TpDex, loop deletion, salt bridge, and s...

Journal of Applied Glycoscience, 2001

Journal of Applied Glycoscience, 2002

Agricultural and biological chemistry

Journal of Biological Chemistry

T w o lectins (HOL-I and HOL-11) were isolated from the marine sponge Halichondria okadai by affi... more T w o lectins (HOL-I and HOL-11) were isolated from the marine sponge Halichondria okadai by affinity chromatography on a bovine submaxillary mucin (BSM)-Toyopearl and an acid-treated Sepharose 4B columns, respectively. In hemagglutination inhibition assays, GlcNAc, GalNAc, and their methyl glycosides were the most potent inhibitors among the monosaccharides tested against the HOL-I-mediated hemagglutination, suggesting that HOL-I can especially recognize the N-acetyl groups of the sugars. This N-acetyl specificity was supported by 'H NMR analyses; the highest field-shifts of the signal of the N-acetyl group among all the signals in Me PGlcNAc were observed in 'H NMR spectra of mixtures of HOL-I and the sugar. Among the oligosaccharides tested, GlcNAcpl+4(GlcNAc~l+2)Manc~1-O(CH~)~ CHs was the most potent inhibitor, and the inhibitory potency of the oligosaccharide was z4 times greater than those of GlcNAc and GalNAc. On the other hand, N-acetyllactosamine (Galpl-rlGlcNAc) and its analogs were the strongest inhibitors toward HOL-11-induced hemagglutination. The agglutination was completely inert to Galpl+3GlcNAc, GalPl-rGGlcNAc, Galpl-SGalNAc, GalPl+lGalNAc, and Galpl+GGalNAc. Furthermore, HOL-I1 exhibited no binding ability to BSM, asialo-BSM, fetuin, asialofetuin, a,-acid glycoprotein, and human transferrin. These results indicate that HOL-I1 strictly recognizes simple Galpl-r4GlcNAc unit. Many lectins have been isolated from various sources. However, information is relatively sparse concerning lectins from marine origins as compared with the large variety of purified lectins of the other sources. It is well known that marine organisms produce unique low molecular compounds possessing unique structures and biological activities. It is also possible that novel lectins having unique properties will be found from the organisms.

Phytochemistry

Two lectins, PAL-I and PAL-II, were isolated from the mushroom Phaeolepiota aurea by affinity chr... more Two lectins, PAL-I and PAL-II, were isolated from the mushroom Phaeolepiota aurea by affinity chromatography on acid-treated Sepharose CL-4B followed by reverse-phase FPLC on ProRPC. Both of the lectins were tetramers of 16 kDa subunits. The lectins had little ...

Journal of Biochemistry

The substrate specificity of honeybee alpha-glucosidase I, a monomeric enzyme was kinetically inv... more The substrate specificity of honeybee alpha-glucosidase I, a monomeric enzyme was kinetically investigated. Unusual kinetic features were observed in the cleavage reactions of sucrose, maltose, p-nitrophenyl alpha-glucoside, phenyl alpha-glucoside, turanose, and maltodextrin (DP = 13). At relatively high substrate concentrations, the velocities of liberation of fructose from sucrose, glucose from maltose, p-nitrophenol from p-nitrophenyl alpha-glucoside, and phenol from phenyl alpha-glucoside were accelerated, and so the Lineweaver-Burk plots were convex, indicating negative kinetic cooperativity: the Hill coefficients were calculated to be 0.50, 0.64, 0.50, and 0.67 for sucrose, maltose, p-nitrophenyl alpha-glucoside, and phenyl alpha-glucoside, respectively. For the degradation of turanose and maltodextrin, the enzyme showed a sigmoidal curve in v versus s plots and thus catalyzed the reaction with positive kinetic cooperativity. The Lineweaver-Burk plots were concave and the Hill coefficients were 1.2 and 1.5 for turanose and maltodextrin, respectively. These unique properties cannot be interpreted by the reaction mechanism that Huber and Thompson proposed: (1973) Biochemistry 12, 4011-4020. The rate parameters for the hydrolysis of sucrose, maltose, p-nitrophenyl alpha-glucoside and phenyl alpha-glucoside were estimated by extrapolating the linear part of the Lineweaver-Burk plots at low substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

An isomalto-dextranase from Arthrobacter globiformis T6 was kinetically elucidated to be capa ble... more An isomalto-dextranase from Arthrobacter globiformis T6 was kinetically elucidated to be capa ble of splitting ƒ¿-1,4-glucosidic linkage of panose as well as ƒ¿-1,6-glucosidic linkage of isomalt

Plant Science

We found P-19.5 protein that preferentially accumulated in cell clusters and globular embryos at ... more We found P-19.5 protein that preferentially accumulated in cell clusters and globular embryos at the early stage of carrot somatic embryogenesis. P-19.5 protein was isolated from 9-day-old cell clusters with SDS-PAGE, and then determined the amino acid sequences of a N-terminal peptide and some internal peptides. Using each primer for these peptides, the full-length sequence of cDNA encoding P-19.5 protein was determined by analyzing 3′-RACE and 5′-RACE products. We regard P-19.5 protein as an allergen-like protein because the P-19.5 protein sequence predicted from cDNA was homologous to celery major allergen (75%), carrot major allergen (70–72%), Pimpinella brachycarpa pathogenesis-related protein (71%), and parsley pathogenesis-related protein (63%). We found concomitantly two separate cDNAs (P-16 and P-19.5 H cDNAs) during analyzing P-19.5 cDNA: P-16 cDNA was cloned and isolated from P-16 protein that was recognized against P-19.5 protein antiserum, and P-19.5 H cDNA that had hom...

Agricultural and Biological Chemistry, 1991

Asp. niger a-glucosidase wascrystallized in ammonium sulfate solution after chromatographies on D... more Asp. niger a-glucosidase wascrystallized in ammonium sulfate solution after chromatographies on DEAE-Sepharose CL-6Band TOYOPEARL HW-55columns. The crystalline a-glucosidase, which was a glycoprotein containing 25.5% carbohydrate as glucose, gave a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be about 12.5 x 104 by SDS-disc gel electrophoresis. However, the crystalline enzyme consists of two components (M.W. about 3.3 x 104 and 9.8 x 104, respectively) separable only by reverse-phase HPLCcausing irreversible inactivation. The optimumpH was 4.3. The enzymealso hydrolyzed a-glucans such as soluble starch and amylose. The ratios of l values (Km values, in parentheses, him of nonreducing terminal) for maltose,

Bioscience Biotechnology and Biochemistry

A novel glucanhydrolase from a mutant of Lipomyces starkeyi ATCC 74054 was purified. The single p... more A novel glucanhydrolase from a mutant of Lipomyces starkeyi ATCC 74054 was purified. The single protein (100 kDa) showed either dextranolytic or amylolytic activity. We referred to the glucanhydrolase as a DXAMase. The DXAMase was produced in a starch medium and it was 3.75-fold more active for hydrolysis of the purified insoluble-glucan of Streptococcus mutans than Penicillium funiculosum dextranase. Aggregation of S. mutans cells with dextran and adherence to glass were eliminated by incubating with the DXAMase. The addition of DXAMase (0.1 IU/ml) to the mutansucrase reaction digest with sucrose reduced the formation of insoluble-glucan about 80%. Also the DXAMase (0.5 IU/ml) removed 80% of the pre-formed sucrose-dependent adherent film. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent.

XXIst International Carbohydrate Symposium 2002, 2002