Biomedical subjects

Haruhide Mori

Publications and source records attributed to Haruhide Mori.

16 recordsLinked to original sources

PubMed2006

Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA.

alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.

Animals↗

PubMed2006

Multiple forms of alpha-glucosidase in rice seeds (Oryza sativa L., var Nipponbare).

Two isoforms of alpha-glucosidases (ONG2-I and ONG2-II) were purified from dry rice seeds (Oryza sativa L., var Nipponbare). Both ONG2-I and ONG2-II were the gene products of ONG2 mRNA expressed in ripening seeds. Each enzyme consisted of two components of 6kDa-peptide and 88kDa-peptide encoded by this order in ONG2 cDNA (ong2), and generated by post-translational proteolysis. The 88kDa-peptide of ONG2-II had 10 additional N-terminal amino acids compared with the 88kDa-peptide of ONG2-I. The peptides between 6kDa and 88kDa components (26 amino acids for ONG2-I and 16 for ONG2-II) were removed by post-translational proteolysis. Proteolysis induced changes in adsorption and degradation of insoluble starch granules. We also obtained three alpha-glucosidase cDNAs (ong1, ong3, and ong4) from ripening seeds. The ONG1, ONG2, and ONG4 genes were situated in distinct locus of rice genome. The transcripts encoding ONG2 and ONG3 were generated by alternative splicing. Members of alpha-glucosidase multigene family are differentially expressed during ripening and germinating stages in rice.

Alternative Splicing↗

PubMed2006

Structural elements to convert Escherichia coli alpha-xylosidase (YicI) into alpha-glucosidase.

Escherichia coli YicI, a member of glycoside hydrolase family (GH) 31, is an alpha-xylosidase, although its amino-acid sequence displays approximately 30% identity with alpha-glucosidases. By comparing the amino-acid sequence of GH 31 enzymes and through structural comparison of the (beta/alpha)(8) barrels of GH 27 and GH 31 enzymes, the amino acids Phe277, Cys307, Phe308, Trp345, Lys414, and beta-->alpha loop 1 of (beta/alpha)(8) barrel of YicI have been identified as elements that might be important for YicI substrate specificity. In attempt to convert YicI into an alpha-glucosidase these elements have been targeted by site-directed mutagenesis. Two mutated YicI, short loop1-enzyme and C307I/F308D, showed higher alpha-glucosidase activity than wild-type YicI. C307I/F308D, which lost alpha-xylosidase activity, was converted into alpha-glucosidase.

Amino Acid Sequence↗

PubMed2006

Structural elements in dextran glucosidase responsible for high specificity to long chain substrate.

Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to alpha-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short beta-->alpha loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar kcat/Km value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer beta-->alpha loop 4 of B. subtilis oligo-1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short beta-->alpha loop 4 were important for high activity to long-chain substrates.

Amino Acid Sequence↗

PubMed2005

Oligosaccharide binding to barley alpha-amylase 1.

Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."

Acarbose↗

PubMed2005

Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley alpha-amylase.

Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite -5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites.

Amylose↗

PubMed2005

Crystallization and preliminary X-ray analysis of alpha-xylosidase from Escherichia coli.

Glycoside hydrolases have been implicated in many biological processes. To date, they have been classified into 93 glycoside hydrolase (GH) families based on amino-acid sequence similarity. alpha-Xylosidase from Escherichia coli belongs to GH family 31 and catalyzes the release of alpha-xylose from the non-reducing terminal side of alpha-xyloside. Single crystals of alpha-xylosidase have been grown by vapour diffusion at 293 K from 10%(w/v) PEG 20K, 2%(v/v) 2-propanol, 2%(v/v) glycerol and 0.1 M 2-morpholinoethanesulfonic acid pH 5.5. These crystals belong to space group P2(1)2(1)2(1) and X-ray diffraction data were collected to a resolution of 2.75 A. Crystals of selenomethionyl-substituted alpha-xylosidase were also obtained, which diffracted to at least 3.0 A. Based on the value of VM, the asymmetric unit in these crystals was assumed to contain six molecules.

Escherichia coli↗

PubMed2005

Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase.

A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k(cat)/K(m)) for maltooligosaccharides, compared with that for soluble starch. The product anomer was alpha-glucose, differing from glucoamylase as a beta-glucose producing enzyme. These findings are striking characteristics of alpha-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian alpha-glucosidases of alpha-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of alpha-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical alpha-glucosidases, and not glucoamylase.

Catalytic Domain↗

PubMed2004

Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage.

alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.

Acremonium↗

PubMed2004

Overexpression and characterization of two unknown proteins, YicI and YihQ, originated from Escherichia coli.

The proteins encoded in the yicI and yihQ gene of Escherichia coli have similarities in the amino acid sequences to glycoside hydrolase family 31 enzymes, but they have not been detected as the active enzymes. The functions of the two proteins have been first clarified in this study. Recombinant YicI and YihQ produced in E. coli were purified and characterized. YicI has the activity of alpha-xylosidase. YicI existing as a hexamer shows optimal pH at 7.0 and is stable in the pH range of 4.7-10.1 with incubation for 24h at 4 degrees C and also is stable up to 47 degrees C with incubation for 15 min. The enzyme shows higher activity against alpha-xylosyl fluoride, isoprimeverose (6-O-alpha-xylopyranosyl-glucopyranose), and alpha-xyloside in xyloglucan oligosaccharides. The alpha-xylosidase catalyzes the transfer of alpha-xylosyl residue from alpha-xyloside to xylose, glucose, mannose, fructose, maltose, isomaltose, nigerose, kojibiose, sucrose, and trehalose. YihQ exhibits the hydrolysis activity against alpha-glucosyl fluoride, and so is an alpha-glucosidase, although the natural substrates, such as alpha-glucobioses, are scarcely hydrolyzed. alpha-Glucosidase has been found for the first time in E. coli.

Amino Acid Sequence↗

PubMed2004

Localization of alpha-glucosidases I, II, and III in organs of European honeybees, Apis mellifera L., and the origin of alpha-glucosidase in honey.

Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an alpha-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey alpha-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.

Amino Acid Sequence↗

PubMed2003

Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus.

Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N-terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18-residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68,993 Da. ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o-diphenols and to exhibit temperature-dependency of o-diphenolase activity like a psychrophilic enzyme.

Amino Acid Sequence↗

PubMed2003

Purification, characterization, and sequence analysis of two alpha-amylase isoforms from azuki bean, Vigna angularis, showing different affinity towards beta-cyclodextrin sepharose.

Two alpha-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a beta-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl2, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17): kcat, 71.8 and 55.5 s(-1), Km, 0.113 and 0.097 mg/ml; for blocked 4-nitrophenyl alpha-D-maltoheptaoside: kcat, 62.4 and 85.3 s(-1), Km, 0.22 and 0.37 mM, respectively. Primary structures of the two enzymes were analyzed by N-terminal sequencing, cDNA cloning, and MALDI-TOF mass spectrometry, implying that the two enzymes have the same peptide. The results indicated that the low affinity of VAAmy1 towards beta-cyclodextrin Sepharose was due to some modification on/near carbohydrate binding site in the limited sequence regions, resulting in higher molecular weight.

Amino Acid Sequence↗

PubMed2002

Barley alpha-amylase Met53 situated at the high-affinity subsite -2 belongs to a substrate binding motif in the beta-->alpha loop 2 of the catalytic (beta/alpha)8-barrel and is critical for activity and substrate specificity.

Met53 in barley alpha-amylase 1 (AMY1) is situated at the high-affinity subsite -2. While Met53 is unique to plant alpha-amylases, the adjacent Tyr52 stacks onto substrate at subsite -1 and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in beta-->alpha loop 2 of the catalytic (beta/alpha)8-barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53. Compared to wild-type, Met53Glu/Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly 76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13. Towards amylose DP17 (degree of polymerization = 17) and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside the activity (kcat/Km) of all mutants was reduced to 5.5-0.01 and 1.7-0.02% of wild-type, respectively. Km increased up to 20-fold for these soluble substrates and the attack on glucosidic linkages in 4-nitrophenyl alpha-d-maltohexaoside (PNPG6) and PNPG5 was determined by action pattern analysis to shift to be closer to the nonreducing end. This indicated that side chain replacement at subsite -2 weakened substrate glycon moiety contacts. Thus whereas all mutants produced mainly PNPG2 from PNPG6 and similar amounts of PNPG2 and PNPG3 accounting for 85% of the products from PNPG5, wild-type released 4-nitrophenol from PNPG6 and PNPG and PNPG2 in equal amounts from PNPG5. Met53Trp affected the action pattern on PNPG7, which was highly unusual for AMY1 subsite mutants. It was also the sole mutant to catalyze substantial transglycosylation - promoted probably by slow substrate hydrolysis - to produce up to maltoundecaose from PNPG6.

Acarbose↗

PubMed2002

Identification of essential ionizable groups and evaluation of subsite affinities in the active site of beta-D-glucosidase F1 from a Streptomyces sp.

Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of beta-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of beta-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and Km values on pH, when the substrate concentration was sufficiently lower than Km, gave the values of 4.1 and 7.2 for the ionization constants, pKe1 and pKe2 of essential ionizable groups 1 and 2 of the free enzyme, respectively. When the dielectric constant of the reaction mixture was decreased in the presence of 10% methanol, the pKe1 and pKe2, values shifted to higher, to +0.60 and +0.35 pH unit, respectively. The findings supported the notion that the essential ionizable groups of the enzyme were a carboxylate group (-COO-, the group 1) and a carboxyl group (-COOH, the group 2). The subsite affinities Ai's in the active site were evaluated on the basis of the rate parameters of laminarioligosaccharides. Subsites 1 and 2 having positive Ai values (A1 was 1.10 kcal/mol and A2 was 4.98 kcal/mol) were considered to probably facilitate the binding of the substrate to the active site. However, the subsites 3 and 4 showed negative Ai values (A3 was -0.21 kcal/mol and A4 was -2.8 kcal/mol).

Algorithms↗

PubMed2002

Alpha-glucosidase mutant catalyzes "alpha-glycosynthase"-type reaction.

Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe alpha-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an alpha-glucosidic linkage from beta-glucosyl fluoride and 4-nitrophenyl (PNP) alpha-glucoside to produce two kinds of PNP alpha-diglucosides, alpha-isomaltoside and alpha-maltoside. The two products were not hydrolyzed by D481G, giving 41 and 29% yields of PNP alpha-isomaltoside and alpha-maltoside, respectively. PNP monoglycosides, such as alpha-xyloside, alpha-mannoside, or beta-glucoside, acted as the substrate, but PNP alpha-galactoside and maltose could not. No detectable product was observed in the combination of alpha-glucosyl fluoride and PNP alpha-glucoside. This study is the first report on an "alpha-glycosynthase"-type reaction to form an alpha-glycosidic linkage.

Amino Acid Substitution↗