Bunzo Mikami

Kyoto University, Kioto, Kyōto, Japan

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Publications (229)667.25 Total impact

  • Yo Sonoda · Kimihiko Mizutani · Bunzo Mikami ·
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    ABSTRACT: Spo0M is a sporulation-control protein that is thought to play an essential role in the early stage of endospore formation. While little is known about the functions of Spo0M, a recent phylogenetic study suggests that, based on its amino-acid sequence, Spo0M might belong to the arrestin clan. The crystal structure of the Spo0M protein was determined at a resolution of 2.3 Å. Ten amino acids at the end of the N-terminus were removed to improve the thermal stability of the purified Spo0M protein and the crystal structure of Spo0M was determined by SAD. Spo0M has a well conserved N-terminal domain with an arrestin-like fold, which consists of a β-strand sandwich structure. Surprisingly, the C-terminal domain of Spo0M, which has no structural homology to arrestin-clan proteins, bears significant structural similarity to the FP domain of the human PI31 protein. In addition, Spo0M harbours a potential polar-core structure connecting the N- and C-terminal domains with several salt bridges, as seen in the crystal structures of arrestin and VPS26. The structure reported here constitutes the first structural information on a bacterial protein that shares significant structural homology to members of the arrestin clan and the FP domain.
    Acta Crystallographica Section F: Structural Biology Communications 12/2015; 71(12). DOI:10.1107/S2053230X15020919
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    ABSTRACT: The selection of correct metal ions with high fidelity against competing cellular cations is crucial for the function of many metalloenzymes; however, the understanding of the principles that govern metal selectivity is still incomplete. In this study, the crystal structure of the Tm1162 protein from Thermotoga maritima , a metallo-β-lactamase, is reported. Several crystal structures of wild-type Tm1162 and its mutants were solved. Homologues of Tm1162 are widely distributed in bacteria and archaea, including several human pathogens. The monomer possesses an αβ/βα fold, with the core β-strands having the β-sheet sandwich structure common to the metallo-β-lactamase superfamily. Tm1162 exists as a trimer in the crystal and this trimeric unit is likely to be present in solution. In the trimer, three active sites reside at the interface between subunits, suggesting that the oligomeric assembly is crucial for catalysis. A new type of structurally encoded heterodinuclear site has been identified by confirming the identity of nickel-containing heteronuclear sites in Tm1162 via X-ray absorption spectroscopy and anomalous difference Fourier maps. The second coordination sphere, including His8 and Glu73, maintains the side-chain orientations of histidines and stabilizes the metal-binding site. Nickel coordination was crucial for the oligomerization of Tm1162. The nickel-dependent and manganese-dependent β-lactamase and phosphodiesterase activities of Tm1162 have also been characterized.
    Acta Crystallographica Section D Biological Crystallography 10/2015; 71(Pt 10):2054-2065. DOI:10.1107/S1399004715014807 · 2.67 Impact Factor
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    ABSTRACT: The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Structure 07/2015; 23(9). DOI:10.1016/j.str.2015.06.021 · 5.62 Impact Factor
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    ABSTRACT: Moloney murine leukemia virus reverse transcriptase (MMLV RT) contains fingers, palm, thumb, and connection subdomains as well as an RNase H domain. The DNA polymerase active site resides in the palm subdomain, and the RNase H active site is located in the RNase H domain. The RNase H domain contains a positively charged α-helix called the C helix (H(594)GEIYRRR(601)), that is thought to be involved in substrate recognition. In this study, we expressed three versions of the RNase H domain in Escherichia coli, the wild-type domain (WT) (residues Ile498-Leu671) and two variants that lack the regions containing the C helix (Ile593-Leu603 and Gly595-Thr605, which we called ΔC1 and ΔC2, respectively) with a strep-tag at the N-terminus and a deca-histidine tag at the C-terminus. These peptides were purified from the cells by anion-exchange, Ni(2+) affinity, and Strep-Tactin affinity column chromatography, and then the tags were removed by proteolysis. In an RNase H assay using a 25-bp RNA-DNA heteroduplex, WT, ΔC1, and ΔC2 produced RNA fragments ranging from 7 to 16 nucleotides (nt) whereas the full-length MMLV RT (Thr24-Leu671) produced 14-20-nt RNA fragments, suggesting that elimination of the fingers, palm, thumb, and connection subdomains affects the binding of the RNase H domain to the RNA-DNA heteroduplex. The activity levels of WT, ΔC1, and ΔC2 were estimated to be 1%, 0.01%, and 0.01% of full-length MMLV RT activity, indicating that the C helix is important, but not critical, for the activity of the isolated RNase H domain. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 05/2015; 113. DOI:10.1016/j.pep.2015.04.012 · 1.70 Impact Factor
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    ABSTRACT: β-Conglycinin is a major seed storage protein in soybeans, which are an important source of protein. The major subunits (α, α′ and β) of β-conglycinin are sorted to protein-storage vacuoles in seed cells. Vacuolar sorting receptor (VSR) is an integral membrane protein that recognizes the sorting determinant of vacuolar proteins, including β-conglycinin, and regulates their sorting process. Vacuolar sorting determinants of the α′ and β subunits of β-conglycinin exist in their C-terminal peptides. Here, the preliminary X-ray diffraction analysis of the binding domain of soybean VSR crystallized with the peptide responsible for the sorting determinant in β-conglycinin is reported. X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to space group P 3 1 21, with unit-cell parameters a = b = 116.4, c = 86.1 Å.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2015; 71(Pt 2):132-5. DOI:10.1107/S2053230X14027484 · 0.53 Impact Factor
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    ABSTRACT: Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase (UGL). Genes coding for glycosaminoglycans-acting lyase, UGL, and phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-L-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-D-gluconate via 3-deoxy-D-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-D-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, while KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by X-ray crystallography. Distinct from KduI β-barrels, DhuI adopts an α/β/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of glycosaminoglycans-derived unsaturated uronic acids by isomerase and dehydrogenase. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 01/2015; 290(10). DOI:10.1074/jbc.M114.604546 · 4.57 Impact Factor
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    ABSTRACT: 5-Formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase (FHMPCDH) from Mesorhizobium loti is the fifth enzyme in degradation pathway I for pyridoxine. The enzyme catalyzes a dismutation reaction: the oxidation of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) to 3-hydroxy-2-methylpyridine 4,5-dicarboxylic acid with NAD(+) and reduction of FHMPC to 4-pyridoxic acid with NADH. FHMPCDH belongs to the l-3-hydroxyacyl-CoA dehydrogenase (HAD) family. The crystal structure was determined by molecular replacement and refined to a resolution of 1.55Å (R-factor of 16.4%, Rfree=19.4%). There were two monomers in the asymmetric unit. The overall structure of the monomer consisted of N- and C-terminal domains connected by a short linker loop. The monomer was similar to members of the HAD family (RMSD=1.9Å). The active site was located between the domains and highly conserved to that of human heart l-3-hydroxyacyl-CoA dehydrogenase (HhHAD). His-Glu catalytic dyad, a serine and two asparagine residues of HhHAD were conserved. Ser116, His137 and Glu149 in FHMPC dehydrogenase are connected by a hydrogen bonding network forming a catalytic triad. The functions of the active residues in the reaction mechanism are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.
    Biochemical and Biophysical Research Communications 11/2014; 456(1). DOI:10.1016/j.bbrc.2014.11.028 · 2.30 Impact Factor
  • Tetsuya Masuda · Bunzo Mikami · Fumito Tani ·
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    ABSTRACT: Thaumatin, an intensely sweet-tasting protein used as a sweetener, elicits a sweet taste at 50 nM. Although two major variants designated thaumatin I and thaumatin II exist in plants, there have been few dedicated thaumatin II structural studies and, to date, data beyond atomic resolution had not been obtained. To identify the detailed structural properties explaining why thaumatin elicits a sweet taste, the structure of recombinant thaumatin II was determined at the resolution of 0.99 Å. Atomic resolution structural analysis with riding hydrogen atoms illustrated the differences in the direction of the side-chains more precisely and the electron density maps of the C-terminal regions were markedly improved. Though it had been suggested that the three consecutive glycine residues (G142-G143-G144) have highly flexible conformations, G143, the central glycine residue was successfully modelled in two conformations for the first time. Furthermore, the side chain r.m.s.d. values for two residues (R67 and R82) critical for sweetness exhibited substantially higher values, suggesting that these residues are highly disordered. These results demonstrated that the flexible conformations in two critical residues favoring their interaction with sweet taste receptors are prominent features of the intensely sweet taste of thaumatin.
    Biochimie 11/2014; 106C. DOI:10.1016/j.biochi.2014.07.016 · 2.96 Impact Factor
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    ABSTRACT: The alginate-assimilating bacterium, Sphingomonas sp. strain A1, degrades the polysaccharide to monosaccharides through four alginate lyases reactions. The resultant monosaccharide, which is nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), is further metabolized to 2-keto-3-deoxy-D-gluconate by NADPH-dependent reductase A1-R in the short-chain dehydrogenase/reductase (SDR) family. A1-R-deficient cells produced another DEH reductase, designated A1-R', with a preference for NADH. Here we show the identification of a novel NADH-dependent DEH reductase A1-R' in strain A1, structure determination of A1-R' by X-ray crystallography, and structure-based conversion of a coenzyme requirement in SDR enzymes, A1-R and A1-R'. A1-R' was purified from strain A1 cells and enzymatically characterized. Except for coenzyme requirement, there was no significant difference in enzyme characteristics between A1-R and A1-R'. Crystal structures of A1-R' and A1-R'/NAD+ complex were determined at 1.8 and 2.7 Å resolutions, respectively. Because of a 64% sequence identity, overall structures of A1-R' and A1-R were similar, although a difference in the coenzyme-binding site (particularly nucleoside ribose 2' region) was observed. Distinct from A1-R, A1-R' included a negatively charged, shallower binding site. These differences were caused by amino acid residues on the two loops around the site. The A1-R' mutant with the two A1-R-typed loops maintained potent enzyme activity with specificity for NADPH rather than NADH, demonstrating that the two loops determine the coenzyme requirement and loop exchange is a promising method for conversion of coenzyme requirement in the SDR family.
    Journal of Biological Chemistry 10/2014; 289(48). DOI:10.1074/jbc.M114.585661 · 4.57 Impact Factor
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    Taro Masuda · Guanghua Zhao · Bunzo Mikami ·
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    ABSTRACT: Chitinase hydrolyzes the β-1,4-glycosidic bond in chitin. In higher plants, this enzyme has been regarded as a pathogenesis-related protein. Recently, we identified a class III chitinase, which functions as a calcium storage protein in pomegranate (Punica granatum) seed (PSC, pomegranate seed chitinase). Here, we solved a crystal structure of PSC at 1.6 Å resolution. Although its overall structure, including the structure of catalytic site and non-proline cis-peptides, was closely similar to those of other class III chitinases, PSC had some unique structural characteristics. First, there were some metal-binding sites with coordinated water molecules on the surface of PSC. Second, many unconserved aspartate residues were present in the PSC sequence which rendered the surface of PSC negatively charged. This acidic electrostatic property is in contrast to that of hevamine, well-characterized plant class III chitinase, which has rather a positively charged surface. Thus, the crystal structure provides a clue for metal association property of PSC.
    Bioscience Biotechnology and Biochemistry 09/2014; 79(1):1-6. DOI:10.1080/09168451.2014.962475 · 1.06 Impact Factor
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    ABSTRACT: Elucidation of the three-dimensional structure of biomolecules is of great importance because the three-dimensional structure is closely related to biological functions. X-ray single-crystal analysis is powerful method to analyze the structure, but it is sometimes difficult to grow a crystal sufficiently large for conventional or even synchrotron single-crystal X-ray measurement. We recently reported on a magnetically oriented microcrystal array (MOMA) [1] that is a composite in which microcrystals are aligned three-dimensionally in polymer matrix. Microcrystals are suspended in an ultraviolet-curable monomer and rotated non-uniformly in a static magnetic field to achieve three dimensional crystal alignment. Then, the monomer is photopolymerized to maintain the achieved alignment. We have successfully demonstrated that X-ray single crystal structure determinations through MOMA are possible for low molecular weight compounds [2] as well as protein. [3] However, the method with MOMA has two drawbacks: (i) the sample microcrystals cannot be recovered from a MOMA, which is especially serious problem in case of proteins, and (ii) the alignment is deteriorated during the consolidation process, causing low resolution. In this study, we attempt to solve these problems. First, we use a water-soluble sol as microcrystalline media and consolidate the alignment by gelation, which makes the recovery of microcrystals possible. Second, a magnetically oriented microcrystal suspension (MOMS) is used for in-situ X-ray diffraction measurement, which makes the sample recovery possible and enhances the resolution. We use lysozyme as a model protein for both cases. The in-situ method with in-house X-ray diffractometer gave diffraction spots about 3.0 Å resolutions. We plan to perform the same experiment at SPring-8.
    Acta Crystallographica Section A: Foundations and Advances 08/2014; 70(a1):C349-C349. DOI:10.1107/S2053273314096508
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    ABSTRACT: 4-Pyridoxolactonase from Mesorhizobium loti catalyzes the zinc-dependent lactone-ring hydrolysis of 4-pyridoxolactone (4PAL) to 4-pyridoxic acid (4PA) in vitamin B 6 degradation pathway I. The crystal structures of 4-pyridoxolactonase and its complex with 5-pyridoxolactone (5PAL; the competitive inhibitor) were determined. The overall structure was an αβ/βα sandwich fold, and two zinc ions were coordinated. This strongly suggested that the enzyme belongs to subclass B3 of the class B β-lactamases. In the complex structure, the carbonyl group of 5PAL pointed away from the active site, revealing why it acts as a competitive inhibitor. Based on docking simulation with 4PAL, 4PA and a reaction intermediate, 4-pyridoxolactonase probably catalyzes the reaction through a subclass B2-like mechanism, not the subclass B3 mechanism.
    Acta Crystallographica Section F: Structural Biology Communications 04/2014; 70(Pt 4):424-32. DOI:10.1107/S2053230X14003926
  • Taro Masuda · Kyosuke Momoji · Takashi Hirata · Bunzo Mikami ·
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    ABSTRACT: Unlabelled: Phenoloxidase (PO), which is classified as a type 3 copper protein, catalyzes the hydroxylation of monophenol to o-diphenol and subsequent oxidation to the corresponding o-quinone. The geometry and coordination environment of the active site of the arthropod PO are very similar to those of the arthropod hemocyanin (Hc). However, unlike the POs, Hc is an oxygen carrier in crustaceans, and does not possess PO activity in general. Recently, we identified a new type of proPO from a crustacean and designated it proPOβ. This enzyme has many characteristics that are rather similar to those of Hc, such as its maturation, localization, and oligomeric state. Here, we determined the crystal structure of proPOβ prepared from the hemolymph of kuruma prawns (Marsupenaeus japonicus) at 1.8-Å resolution. M. japonicus proPOβ forms a homohexamer rather similar to that of arthropod Hc. The geometry of the active copper site in proPOβ is nearly identical to that of arthropod Hc. Furthermore, the well-characterized 'place-holder' phenylalanine is present (Phe72). However, the accessibility to the active site differs in several ways. First, another phenylalanine, which shields the active site by interacting with a copper-coordinated histidine in crustacean Hc, is replaced by valine in the proPOβ structure. Second, two tyrosines, Tyr208 and Tyr209, both of which are absent in Hc, show the alternative conformations and form a pathway providing access to the reaction center. Thus, the present crystal structure clarifies the similarities and differences in the activity of two closely related proteins, PO and Hc. Database: Structural data are available in the RSCB protein data bank under the accession number 3WKY. ray crystallography (View interaction).
    FEBS Journal 04/2014; 281(11). DOI:10.1111/febs.12812 · 4.00 Impact Factor
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    ABSTRACT: Pedobacter heparinus (formerly known as Flavobacterium heparinum) is a typical glycosaminoglycan-degrading bacterium that produces three heparin lyases, Hep I, Hep II, and Hep III, which act on heparins with 1-4 glycoside bonds between uronate and amino sugar residues. As different from Hep I and Hep II, Hep III is specific for heparan sulfate. Here we describe the crystal structure of Hep III with active site located in a deep cleft. The X-ray crystallographic structure of Hep III was determined at 2.20 Å resolution using single-wavelength anomalous diffraction. This enzyme comprised an N-terminal α/α-barrel domain and a C-terminal antiparallel β-sheet domain as its basic scaffold. Overall structures of Hep II and Hep III were similar, although Hep III exhibited an open form compared with the closed form of Hep II. Superimposition of Hep III and heparin tetrasaccharide-bound Hep II suggested that an active site of Hep III was located in the deep cleft at the interface between its two domains. Three mutants (N240A, Y294F, and H424A) with mutations at the active site had significantly reduced enzyme activity. This is the first report on the structure-function relationship of P. heparinus Hep III.
    Biochemistry 01/2014; 53(4). DOI:10.1021/bi4012463 · 3.02 Impact Factor
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    ABSTRACT: Extracellular matrix molecules such as glycosaminoglycans (GAGs) are typical targets for some pathogenic bacteria, which allow adherence to host cells. Bacterial polysaccharide lyases depolymerize GAGs in β-elimination reactions, and the resulting unsaturated disaccharides are subsequently degraded to constituent monosaccharides by unsaturated glucuronyl hydrolases (UGLs). UGL substrates are classified as 1,3- and 1,4-types based on the glycoside bonds. Unsaturated chondroitin and heparin disaccharides are typical members of 1,3- and 1,4-types, respectively. Here we show the reaction modes of bacterial UGLs with unsaturated heparin disaccharides by X-ray crystallography, docking simulation, and site-directed mutagenesis. Although streptococcal and bacillus UGLs were active on unsaturated heparin disaccharides, those preferred 1,3- rather than 1,4-type substrates. The genome of GAG-degrading Pedobacter heparinus encodes 13 UGLs. Of these, Phep_2830 is known to be specific for unsaturated heparin disaccharides. The crystal structure of Phep_2830 was determined at 1.35 Å resolution. In comparison with structures of streptococcal and bacillus UGLs, a pocket-like structure and lid loop at subsite +1 are characteristic of Phep_2830. Docking simulations of Phep_2830 with unsaturated heparin disaccharides demonstrated that the direction of substrate pyranose rings differs from that in unsaturated chondroitin disaccharides. Acetyl groups of unsaturated heparin disaccharides are well accommodated in the pocket at subsite +1, and aromatic residues of the lid loop are required for stacking interactions with substrates. Thus, site-directed mutations of the pocket and lid loop led to significantly reduced enzyme activity, suggesting that the pocket-like structure and lid loop are involved in the recognition of 1,4-type substrates by UGLs.
    Journal of Biological Chemistry 01/2014; 289(8). DOI:10.1074/jbc.M113.522573 · 4.57 Impact Factor

  • Acta Crystallographica Section A Foundations of Crystallography 08/2013; 69(a1):s330-s330. DOI:10.1107/S0108767313097146 · 2.31 Impact Factor
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    ABSTRACT: Glycinin is one of the most abundant storage-protein molecules in soybean seeds and is composed of five subunits (A1aB1b, A1bB2, A2B1a, A3B4 and A5A4B3). A1bB2 was purified from a mutant soybean cultivar containing glycinin composed of only A5A4B3 and A1bB2. At 281 K the protein formed hexagonal, rectangular and rod-shaped crystals in the first [0.1 M imidazole pH 8.0, 0.2 M MgCl2, 35%(v/v) MPD], second [0.1 M sodium citrate pH 5.6, 0.2 M ammonium acetate, 30%(v/v) MPD] and third (0.1 M phosphate-citrate pH 4.2, 2.0 M ammonium sulfate) crystallization conditions, respectively. X-ray diffraction data were collected to resolutions of 1.85, 1.85 and 2.5 Å from crystals of the three different shapes. The crystals belonged to space groups P6322, P21 and P1, with unit-cell parameters a = b = 143.60, c = 84.54 Å, a = 114.54, b = 105.82, c = 116.67 Å, β = 94.99° and a = 94.45, b = 94.96, c = 100.66 Å, α = 107.02, β = 108.44, γ = 110.71°, respectively. One, six and six subunits of A1bB2 were estimated to be present in the respective asymmetric units. The three-dimensional structure of the A1bB2 hexamer is currently being determined.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2013; 69(Pt 8):937-941. DOI:10.1107/S1744309113019684 · 0.53 Impact Factor
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    ABSTRACT: Pyridoxine 4-oxidase (PNOX) from Mesorhizobium loti is a monomeric glucose-methanol-choline (GMC) oxidoreductase family enzyme, catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal, and is the first enzyme in pathway I for degradation of PN. The tertiary structures of PNOX with a C-terminal His6-tag and PNOX-pyridoxamine (PM) complex were determined at 2.2Å and at 2.1Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1Å from the N4' atom of PM. The activities of His460Ala and His462Ala mutant PNOXs were very low, and 460Ala/His462Ala double mutant PNOX exhibited no activity. His462 may act as a general base for abstraction of a proton from the 4'-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4' atom in PM is located at 3.2Å, and the hydride ion from the C4' atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase shows that Pro504 in PNOX corresponds to Asn or His of the conserved His-Asn or His-His pair in other GMC oxidoreductases. The function of the novel proline residue was discussed.
    Biochimica et Biophysica Acta 03/2013; 1834(6). DOI:10.1016/j.bbapap.2013.03.004 · 4.66 Impact Factor
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    ABSTRACT: Amaranth is a crop known for its high quality proteins. 11S Globulin is one of the most abundant and important storage proteins of the amaranth grain. Here, we report the crystal structure of amaranth 11S proglobulin at a final resolution of 2.28 Å. It belonged to the space group P6(3) with cell dimensions a=b=96.6, c=75.0 Å. It contains one asymmetric unit consisting of 372 residues and 100 water molecules. Disordered regions in the model approximately correspond to the variable regions of the 11S globulins. The structure has an extended α-helix and β-barrel domains at both N-terminal and C-terminal regions, which are characteristic of the 11S and 7S globulins. The three dimensional structure suggests that its high thermal stability is due to the cumulative effects of many factors and its good emulsifying property depended on the balance between its surface hydrophobicity and hydrophilicity.
    Food Chemistry 11/2012; 135(2):819-26. DOI:10.1016/j.foodchem.2012.04.135 · 3.39 Impact Factor
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    ABSTRACT: β-1,4-Mannanase (EC catalyzes the hydrolysis of β-1,4-glycosidic bonds within mannan, a major constituent group of the hemicelluloses. Bivalves and gastropods possess β-1,4-mannanase and may degrade mannan in seaweed and/or phytoplankton to obtain carbon and energy using the secreted enzymes in their digestive systems. In the present study, the crystal structure of AkMan, a gastropod β-1,4-mannanase prepared from the common sea hare Aplysia kurodai, was determined at 1.05 Å resolution. This is the first report of the three-dimensional structure of a gastropod β-1,4-mannanase. The structure was compared with bivalve β-1,4-mannanase and the roles of residues in the catalytic cleft were investigated. No obvious binding residue was found in subsite +1 and the substrate-binding site was exposed to the molecular surface, which may account for the enzymatic properties of mannanases that can digest complex substrates such as glucomannan and branched mannan.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2012; 68(Pt 10):1164-8. DOI:10.1107/S1744309112037074 · 0.53 Impact Factor

Publication Stats

4k Citations
667.25 Total Impact Points


  • 1981-2015
    • Kyoto University
      • • Division of Applied Life Sciences
      • • Division of Agronomy and Horticultural Science
      • • Division of Food Science and Biotechnology
      Kioto, Kyōto, Japan
  • 1993-1994
    • Albert Einstein College of Medicine
      • Department of Biochemistry
      New York, New York, United States