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Tomitake Tsukihara

Publications and source records attributed to Tomitake Tsukihara.

At least 19 recordsLinked to original sources
PubMed2006

Crystal structure of mammalian alpha1,6-fucosyltransferase, FUT8.

Mammalian alpha1,6-fucosyltransferase (FUT8) catalyses the transfer of a fucose residue from a donor substrate, guanosine 5'-diphosphate-beta-L-fucose to the reducing terminal N-acetylglucosamine (GlcNAc) of the core structure of an asparagine-linked oligosaccharide. Alpha1,6-fucosylation, also referred to as core fucosylation, plays an essential role in various pathophysiological events. Our group reported that FUT8 null mice showed severe growth retardation and emphysema-like lung-destruction as a result of the dysfunction of epidermal growth factor and transforming growth factor-beta receptors. To elucidate the molecular basis of FUT8 with respect to pathophysiology, the crystal structure of human FUT8 was determined at 2.6 A resolution. The overall structure of FUT8 was found to consist of three domains: an N-terminal coiled-coil domain, a catalytic domain, and a C-terminal SH3 domain. The catalytic region appears to be similar to GT-B glycosyltransferases rather than GT-A. The C-terminal part of the catalytic domain of FUT8 includes a Rossmann fold with three regions that are conserved in alpha1,6-, alpha1,2-, and protein O-fucosyltransferases. The SH3 domain of FUT8 is similar to other SH3 domain-containing proteins, although the significance of this domain remains to be elucidated. The present findings of FUT8 suggest that the conserved residues in the three conserved regions participate in the Rossmann fold and act as the donor binding site, or in catalysis, thus playing key roles in the fucose-transferring reaction.

Acetylglucosamine↗
PubMed2006

Structure of coenzyme A-disulfide reductase from Staphylococcus aureus at 1.54 A resolution.

Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Staphylococcus aureus; it is maintained in the reduced state by coenzyme A-disulfide reductase (CoADR), a homodimeric enzyme similar to NADH peroxidase but containing a novel Cys43-SSCoA redox center. The crystal structure of S. aureus CoADR has been solved using multiwavelength anomalous dispersion data and refined at a resolution of 1.54 A. The resulting electron density maps define the Cys43-SSCoA disulfide conformation, with Cys43-S(gamma) located at the flavin si face, 3.2 A from FAD-C4aF, and the CoAS- moiety lying in an extended conformation within a cleft at the dimer interface. A well-ordered chloride ion is positioned adjacent to the Cys43-SSCoA disulfide and receives a hydrogen bond from Tyr361'-OH of the complementary subunit, suggesting a role for Tyr361' as an acid-base catalyst during the reduction of CoAS-disulfide. Tyr419'-OH is located 3.2 A from Tyr361'-OH as well and, based on its conservation in known functional CoADRs, also appears to be important for activity. Identification of residues involved in recognition of the CoAS-disulfide substrate and in formation and stabilization of the Cys43-SSCoA redox center has allowed development of a CoAS-binding motif. Bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/disulfide homeostasis.

Amino Acid Sequence↗
PubMed2006

Proton pumping mechanism of bovine heart cytochrome c oxidase.

X-ray structures of bovine heart cytochrome c oxidase at 1.8/1.9 A resolution in the oxidized/reduced states exhibit a redox coupled conformational change of an aspartate located near the intermembrane surface of the enzyme. The alteration of the microenvironment of the carboxyl group of this aspartate residue indicates the occurrence of deprotonation upon reduction of the enzyme. The residue is connected with the matrix surface of the enzyme by a hydrogen-bond network that includes heme a via its propionate and formyl groups. These X-ray structures provide evidence that proton pumping occurs through the hydrogen bond network and is driven by the low spin heme. The function of the aspartate is confirmed by mutation of the aspartate to asparagine. Although the amino acid residues of the hydrogen bond network and the structures of the low spin heme peripheral groups are not completely conserved amongst members of the heme-copper terminal oxidase superfamily, the existence of low spin heme and the hydrogen bond network suggests that the low spin heme provides the driving element of the proton-pumping process.

Animals↗
PubMed2006

Reaction mechanism of bovine heart cytochrome c oxidase.

The 1.9 A resolution X-ray structure of the O2 reduction site of bovine heart cytochrome c oxidase in the fully reduced state indicates trigonal planar coordination of CuB by three histidine residues. One of the three histidine residues has a covalent link to a tyrosine residue to ensure retention of the tyrosine at the O2 reduction site. These moieties facilitate a four electron reduction of O2, and prevent formation of active oxygen species. The combination of a redox-coupled conformational change of an aspartate residue (Asp51) located near the intermembrane surface of the enzyme molecule and the existence of a hydrogen bond network connecting Asp51 to the matrix surface suggest that the proton-pumping process is mediated at Asp51. Mutation analyses using a gene expression system of the Asp51-containing enzyme subunit yield results in support of the proposal that Asp51 plays a critical role in the proton pumping process.

Animals↗
PubMed2006

The asymmetric IscA homodimer with an exposed [2Fe-2S] cluster suggests the structural basis of the Fe-S cluster biosynthetic scaffold.

It has been shown that the so-called scaffold proteins are vital in Fe-S cluster biosynthesis by providing an intermediate site for the assembly of Fe-S clusters. However, since no structural information on such scaffold proteins with bound Fe-S cluster intermediates is available, the structural basis of the core of Fe-S cluster biosynthesis remains poorly understood. Here we report the first Fe-S cluster-bound crystal structure of a scaffold protein, IscA, from Thermosynechococcus elongatus, which carries three strictly conserved cysteine residues. Surprisingly, one partially exposed [2Fe-2S] cluster is coordinated by two conformationally distinct IscA protomers, termed alpha and beta, with asymmetric cysteinyl ligation by Cys37, Cys101, Cys103 from alpha and Cys103 from beta. In the crystal, two alphabeta dimers form an unusual domain-swapped tetramer via central domains of beta protomers. Together with additional biochemical data supporting its physiologically relevant configuration, we propose that the unique asymmetric Fe-S cluster coordination and the resulting distinct conformational stabilities of the two IscA protomers are central to the function of IscA-type Fe-S cluster biosynthetic scaffold.

Amino Acid Sequence↗
PubMed2006

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453.

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453 have been solved at 2.01 and 2.37A resolutions, respectively. The results revealed that the binding modes for this inhibitor to MMP-3 and -13 were quite similar. However, subtle comparative differences were observed at the bottom of S1' pockets, which were occupied with the guanidinomethyl moiety of the inhibitor. A remarkable feature of the inhibitor was the deep penetration of its long aliphatic chain into the S1' pocket and exposure of the guanidinomethyl moiety to the solvent.

Amino Acid Sequence↗
PubMed2005

Structure of the central hub of bacteriophage Mu baseplate determined by X-ray crystallography of gp44.

Bacteriophage Mu is a double-stranded DNA phage that consists of an icosahedral head, a contractile tail with baseplate and six tail fibers, similar to the well-studied T-even phages. The baseplate of bacteriophage Mu, which recognizes and attaches to a host cell during infection, consists of at least eight different proteins. The baseplate protein, gp44, is essential for bacteriophage Mu assembly and the generation of viable phages. To investigate the role of gp44 in baseplate assembly and infection, the crystal structure of gp44 was determined at 2.1A resolution by the multiple isomorphous replacement method. The overall structure of the gp44 trimer is similar to that of the T4 phage gp27 trimer, which forms the central hub of the T4 baseplate, although these proteins share very little primary sequence homology. Based on these data, we confirm that gp44 exists as a trimer exhibiting a hub-like structure with an inner diameter of 25A through which DNA can presumably pass during infection. The molecular surface of the gp44 trimer that abuts the host cell membrane is positively charged, and it is likely that Mu phage interacts with the membrane through electrostatic interactions mediated by gp44.

Amino Acid Sequence↗
PubMed2005

Absolute configuration of the hydroxyfarnesylethyl group of haem A, determined by X-ray structural analysis of bovine heart cytochrome c oxidase using methods applicable at 2.8 Angstrom resolution.

The absolute configuration of haem A, the prosthetic group of cytochrome c oxidase, was determined to be S by analysis of the bond angles surrounding the chiral centre of haem A after refinement with X-PLOR starting from respective initial structures with R and S configurations under absolute configuration constraints at 1.8 Angstrom resolution. The same result was obtained by refinement at 1.8 Angstrom resolution without the absolute configuration constraints. Both of these methods were applicable down to a resolution of about 2.8 Angstrom. The constrained refinement converges more quickly than the unconstrained refinement.

Animals↗
PubMed2005

Conformational Changes in the tryptophan synthase from a hyperthermophile upon alpha2beta2 complex formation: crystal structure of the complex.

The three-dimensional structure of the bifunctional tryptophan synthase alpha(2)beta(2) complex from Pyrococcus furiosus was determined by crystallographic analysis. This crystal structure, with the structures of an alpha subunit monomer and a beta(2) subunit dimer that have already been reported, is the first structural set in which changes in structure that occur upon the association of the individual tryptophan synthase subunits were observed. To elucidate the structural basis of the stimulation of the enzymatic activity of each of the alpha and beta(2) subunits upon alpha(2)beta(2) complex formation, the conformational changes due to complex formation were analyzed in detail compared with the structures of the alpha monomer and beta(2) subunit dimer. The major conformational changes due to complex formation occurred in the region correlated with the catalytic function of the enzyme as follows. (1) Structural changes in the beta subunit were greater than those in the alpha subunit. (2) Large movements of A46 and L165 in the alpha subunit due to complex formation caused a more open conformation favoring the entry of the substrate at the alpha active site. (3) The major changes in the beta subunit were the broadening of a long tunnel through which the alpha subunit product (indole) is transferred to the beta active site and the opening of an entrance at the beta active site. (4) The changes in the conformations of both the alpha and beta subunits due to complex formation contributed to the stabilization of the subunit association, which is critical for the stimulation of the enzymatic activities.

Amino Acid Sequence↗
PubMed2005

Ab initio crystal structure determination of spherical viruses that exhibit a centrosymmetric location in the unit cell.

Ab initio phasing by non-crystallographic symmetry averaging coupled with solvent flattening has previously been used to determine the structure of canine parvovirus. As CPV particles were located at general positions, initial phases were generated with a spherical shell deviating from the centre of symmetry. In many virus crystals, the viral particles are located at positions with a centre of symmetry in the unit cell. Thus, the initial phases calculated with a spherical shell model have a centre of symmetry. The inherent difficulty in structural determination was breaking the centre of symmetry in the initial phase. The centric nature of the initial phases of the rice dwarf virus crystal, however, was successfully broken at low resolution by iteration of the density-modification method described by Tsao et al. (1992). In this study, ab initio phasing was tested for seven viruses ranging from 82 to 344 A in radius. Although the crystal structures of the initial spherical shell models for each virus had a centre of symmetry, the centric natures of the initial phases were successfully broken by non-crystallographic symmetry averaging coupled with solvent flattening at a low resolution; these phases were then successfully extended to high-resolution measurements. A novel procedure of ab initio phasing for spherical virus crystals is proposed.

Crystallography↗
PubMed2005

Crystallization and preliminary X-ray diffraction study of L-lactate oxidase (LOX), R181M mutant, from Aerococcus viridans.

L-Lactate oxidase (LOX) from Aerococcus viridans is a member of the alpha-hydroxyacid oxidase flavoenzyme family. An X-ray crystallographic study of a LOX mutant in which Arg181 is replaced by Met was initiated in order to understand the functions of the conserved amino-acid residues around the FMN in the enzyme active site. LOX-R181M crystals belong to the tetragonal space group I422, with unit-cell parameters a = b = 192.632, c = 200.263 A, alpha = beta = gamma = 90 degrees. There are four monomers in the asymmetric unit. Diffraction data were collected under cryogenic conditions to 2.44 A resolution from LOX-R181M crystals at BL41XU, SPring-8.

Binding Sites↗
PubMed2005

Conformational changes in the alpha-subunit coupled to binding of the beta 2-subunit of tryptophan synthase from Escherichia coli: crystal structure of the tryptophan synthase alpha-subunit alone.

When the tryptophan synthase alpha- and beta(2)-subunits combine to form the alpha(2)beta(2)-complex, the enzymatic activity of each subunit is stimulated by 1-2 orders of magnitude. To elucidate the structural basis of this mutual activation, it is necessary to determine the structures of the alpha- and beta-subunits alone and together with the alpha(2)beta(2)-complex. The crystal structures of the tryptophan synthase alpha(2)beta(2)-complex from Salmonella typhimurium (Stalpha(2)beta(2)-complex) have already been reported. However, the structures of the subunit alone from mesophiles have not yet been determined. The structure of the tryptophan synthase alpha-subunit alone from Escherichia coli (Ecalpha-subunit) was determined by an X-ray crystallographic analysis at 2.3 A, which is the first report on the subunits alone from the mesophiles. The biggest difference between the structures of the Ecalpha-subunit alone and the alpha-subunit in the Stalpha(2)beta(2)-complex (Stalpha-subunit) was as follows. Helix 2' in the Stalpha-subunit, including an active site residue (Asp60), was changed to a flexible loop in the Ecalpha-subunit alone. The conversion of the helix to a loop resulted in the collapse of the correct active site conformation. This region is also an important part for the mutual activation in the Stalpha(2)beta(2)-complex and interaction with the beta-subunit. These results suggest that the formation of helix 2'that is essential for the stimulation of the enzymatic activity of the alpha-subunit is constructed by the induced-fit mode involved in conformational changes upon interaction between the alpha- and beta-subunits. This also confirms the prediction of the conformational changes based on the thermodynamic analysis for the association between the alpha- and beta-subunits.

Amino Acid Sequence↗
PubMed2005

Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer.

The strongly conserved amino acid sequences of the P8 outer capsid proteins of Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) and the distribution of electrostatic potential on the proteins at the interfaces between structural proteins suggested the possibility that P8-trimers of RGDV might bind to the 3-fold symmetrical axes of RDV core particles, with vertical interaction between heterologous P3 and P8 proteins and lateral binding of homologous P8 proteins, thereby allowing formation of the double-layered capsids that are characteristic of viruses that belong to the family Reoviridae. We proved this hypothesis using chimeric virus-like particles composed of the P3 core capsid protein of RDV and the P8 outer capsid protein of RGDV, which were co-expressed in a baculovirus expression system. This is the first report on the molecular biological proof of the mechanism of the assembly of the double-layered capsids with disparate icosahedral lattices.

Amino Acid Sequence↗
PubMed2005

Expression and molecular characterization of spherical particles derived from the genome of the hyperthermophilic euryarchaeote Pyrococcus furiosus.

Spherical particles (SPs) of approximately 30 nm in diameter were found in the hyperthermophilic archaeon Pyrococcus furiosus. The SPs contained no nucleic acid and were composed of a single 39-kDa protein. The amino acid sequences of the amino-terminal and internal fragments were identical to portions of the deduced amino acid sequence of the putative 38.7-kDa protein encoded by the genome of P. furiosus, suggesting that the protein was expressed from the genome of P. furiosus. This possibility was confirmed by the observation that the 38.7-kDa protein expressed in Escherichia coli reacted specifically with the antibody against purified SPs, and it also formed SPs similar to those found in P. furiosus. Of the 345 amino acid residues in the 38.7-kDa protein, the amino-terminal 100 amino acids exhibited strong homology to putative proteins from other species of Pyrococcus, while the remaining 245 carboxy-terminal residues were not significantly homologous to putative proteins from other members of archaea. Thus, the carboxy-terminal region might be the product of a foreign gene that was incorporated relatively recently into the genome of P. furiosus.

Amino Acid Sequence↗
PubMed2004

Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu.

Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 A. The crystals diffract X-rays to at least 2.1 A resolution and are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 A resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.

Bacteriophage mu↗