PubMed Health⌕ Search

Biomedical subjects

Wataru Hashimoto

Publications and source records attributed to Wataru Hashimoto.

At least 37 records · Page 2Linked to original sources
PubMed2005

Proteomics-based identification of outer-membrane proteins responsible for import of macromolecules in Sphingomonas sp. A1: alginate-binding flagellin on the cell surface.

A nonmotile gram-negative bacterium, Sphingomonas sp. A1, directly incorporates macromolecules such as alginate through a "super-channel" consisting of a pit formed on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Here, we demonstrate the proteomics-based identification of cell-surface proteins involved in the formation of the pit and/or import of alginate. Cell-surface proteins were prepared from the outer membrane released as vesicles during the conversion of intact cells to spheroplasts. Seven proteins (p1-p7) with acidic isoelectric points were inducibly expressed in the outer membrane of strain A1 cells grown on alginate and showed significant identity with bacterial cell-surface proteins (p1-p4, TonB-dependent outer-membrane transporter; p5 and p6, flagellin; and p7, lipoprotein). Each mutant with a disruption of the p1-p4 or p6 gene showed significant growth retardation in the alginate medium. Flagellin homologues (p5 and p6) were further analyzed because strain A1 forms no flagellum. p5 was found to be uniformly distributed on the cell surface by immunogold-labeling electron microscopy and to exhibit alginate binding with a nanomolar dissociation constant by a surface plasmon resonance sensor. The cell surface of the p6 gene disruptant differed from that of the wild-type strain A1 in that pit formation was incomplete and cell-surface structures shifted from pleats to networks. These results suggest that, distinct from bacterial flagellins constituting a helical filament of flagella, strain A1 cell-surface flagellin homologues function as receptors for alginate and/or regulators of cell-surface structures.

Alginates↗
PubMed2005

A structural basis for depolymerization of alginate by polysaccharide lyase family-7.

Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection, but much remains unknown. Here, we present the crystal structure at 1.0 A resolution and the results of mutational analysis of Sphingomonas sp. A1 alginate lyase A1-II', which is grouped into the polysaccharide lyase (PL) family-7. The overall structure of A1-II' uses a beta-sandwich fold, and it has a large active cleft covered by two short flexible loops. Comparison with other family PL-7 structures indicated that loop opening is necessary for substrate binding when the catalytic reaction is initiated. In contrast to the disorder in many side-chains on the protein surface, the three adjacent beta-strands at the center of the active cleft are well ordered. This results from hydrogen bond networks and stacking-like associations identical with those in other family PL-7 structures. Disruption of these interactions by site-directed mutagenesis (R146A, E148A, R150A, Q189A, and K280A) makes the protein insoluble or greatly decreases its activity. The A1-II' structure includes two sulfate ions in the active cleft. Ammonium sulfate was a potent inhibitor with a Ki of 2.5 mM, indicating that our structure represents a model of the inhibitory state. Results of mutational analysis and continuous hydrogen bond networks suggest that Arg146, Gln189, His191, and Tyr284 form an active center. Tyr284OH appears particularly crucial to the catalytic reaction, which is supported by sulfate ion binding and the proximity to the C5 and O4 atoms of subsite +1 in the model obtained by energy minimization calculations using tri-mannuronate. The structural basis shown by this study is similar in many respects to that of the family PL-5 enzymes.

Alginates↗
PubMed2005

Crystal structure of Bacillus sp. GL1 xanthan lyase complexed with a substrate: insights into the enzyme reaction mechanism.

Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8 (PL-8), acts exolytically on the side-chains of pentasaccharide-repeating polysaccharide xanthan and cleaves the glycosidic bond between glucuronic acid (GlcUA) and pyruvylated mannose (PyrMan) through a beta-elimination reaction. To clarify the enzyme reaction mechanism, i.e. its substrate recognition and catalytic reaction, we determined crystal structures of a mutant enzyme, N194A, in complexes with the product (PyrMan) and a substrate (pentasacharide) and in a ligand-free form at 1.8, 2.1, and 2.3A resolution. Based on the structures of the mutant in complexes with the product and substrate, we found that xanthan lyase recognized the PyrMan residue at subsite -1 and the GlcUA residue at +1 on the xanthan side-chain and underwent little interaction with the main chain of the polysaccharide. The structure of the mutant-substrate complex also showed that the hydroxyl group of Tyr255 was close to both the C-5 atom of the GlcUA residue and the oxygen atom of the glycosidic bond to be cleaved, suggesting that Tyr255 likely acts as a general base that extracts the proton from C-5 of the GlcUA residue and as a general acid that donates the proton to the glycosidic bond. A structural comparison of catalytic centers of PL-8 lyases indicated that the catalytic reaction mechanism is shared by all members of the family PL-8, while the substrate recognition mechanism differs.

Bacillus↗
PubMed2005

Direct evidence for Sphingomonas sp. A1 periplasmic proteins as macromolecule-binding proteins associated with the ABC transporter: molecular insights into alginate transport in the periplasm.

A Gram-negative bacterium, Sphingomonas sp. A1, has a macromolecule (alginate) import system consisting of a pit on the cell surface and an alginate-specific ATP-binding cassette importer in the inner membrane. Transport of alginate from the pit to the ABC importer is probably mediated by two periplasmic binding protein homologues (AlgQ1 and AlgQ2). Here we describe characteristics of binding of AlgQ1 and AlgQ2 to alginate and its oligosaccharides through surface plasmon resonance biosensor analysis, UV absorption difference spectroscopy, and X-ray crystallography. Both AlgQ1 and AlgQ2 were inducibly expressed in the periplasm of alginate-grown cells of strain A1. Biosensor analysis indicated that both proteins specifically bind alginate with a high degree of polymerization (>100) and that dissociation constants for alginate with an average molecular mass of 26 kDa are 2.3 x 10(-)(7) M for AlgQ1 and 1.5 x 10(-)(7) M for AlgQ2. An in vitro ATPase assay using the membrane complex, including the alginate ABC importer, suggested that both alginate-bound forms of AlgQ1 and AlgQ2 are closely associated with the importer. X-ray crystallography showed that AlgQ1 consisted of two domains separated by a deep cleft that binds alginate oligosaccharides through a conformational change in the two domains. These results directly show that alginate-binding proteins play an important role in the efficient transport of alginate macromolecules with different degrees of polymerization in the periplasm.

ATP-Binding Cassette Transporters↗
PubMed2005

Crystallization and preliminary X-ray analysis of alginate lyases A1-II and A1-II' from Sphingomonas sp. A1.

Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. The alginate lyases A1-II (25 kDa) and A1-II' (25 kDa) from Sphingomonas sp. A1, which belong to polysaccharide lyase family PL-7, exhibit 68% homology in primary structure but have different substrate specificities. To determine clearly the structural basis for substrate recognition in the depolymerization mechanism by alginate lyases, both proteins were crystallized at 293 K using the vapour-diffusion method. A crystal of A1-II belonged to space group P2(1) and diffracted to 2.2 A resolution, with unit-cell parameters a = 51.3, b = 30.1, c = 101.6 A, beta = 100.2 degrees, while a crystal of A1-II' belonged to space group P2(1)2(1)2(1) and diffracted to 1.0 A resolution, with unit-cell parameters a = 34.6, b = 68.5, c = 80.3 A.

Alginates↗
PubMed2005

NAD-binding mode and the significance of intersubunit contact revealed by the crystal structure of Mycobacterium tuberculosis NAD kinase-NAD complex.

NAD kinase is a key enzyme in NADP biosynthesis. We solved the crystal structure of polyphosphate/ATP-NAD kinase from Mycobacterium tuberculosis (Ppnk) complexed with NAD (Ppnk-NAD) at 2.6A resolution using apo-Ppnk structure solved in this work, and revealed the details of the structure and NAD-binding site. Superimposition of tertiary structures of apo-Ppnk and Ppnk-NAD demonstrated a substantial conformational difference in a loop (Ppnk-flexible loop). As a quaternary structure, these Ppnk structures exhibited tetramer as in solution condition. Notably, the Ppnk-flexible loop was involved in the intersubunit contact and probably related to the NAD-binding of the other subunit. Furthermore, the two residues (Asp189, His226) substantially contributed to creating NAD-binding site on the other subunit. The two residues and the residues involved in NAD-binding were conserved. However, residues corresponding to the Ppnk-flexible loop were not conserved, making us to speculate that the Ppnk-flexible loop may be Ppnk-specific.

Amino Acid Sequence↗
PubMed2005

Molecular identification of Sphingomonas sp. A1 alginate lyase (A1-IV') as a member of novel polysaccharide lyase family 15 and implications in alginate lyase evolution.

Sphingomonas sp. A1 (strain A1) produces three endotypes (A1-I [65 kDa], A1-II [25 kDa], and A1-III [40 kDa]) and an exotype (A1-IV [86 kDa]) alginate lyases in cytoplasm. These four enzymes cooperatively depolymerize alginate into constituent monosaccharides. In addition to the genes for these lyases, novel genes encoding hypothetical proteins homologous with A1-IV were found in the genomes of many bacteria including strain A1. One such protein, A1-IV' (90 kDa) of strain A1, was overexpressed in Escherichia coli cells, purified, and characterized. A1-IV' catalyzed the cleavage of glycosidic bonds in alginate through a beta-elimination reaction and released unsaturated di- and trisaccharides as main products, thus indicating that the enzyme is an endotype alginate lyase. A1-IV', which differed from A1-IV in some enzymatic properties, was not expressed in strain A1, suggesting that A1-IV' has no significant role in alginate metabolism. A1-IV' and other A1-IV homologs facilitate the creation of novel polysaccharide lyase family 15 based on their primary structures, implying the evolution route of alginate lyases in family PL-15.

Amino Acid Sequence↗
PubMed2005

Structure and function of bacterial super-biosystem responsible for import and depolymerization of macromolecules.

Generally, when microbes assimilate macromolecules, they incorporate low-molecular-weight products derived from macromolecules through the actions of extracellular degrading enzymes. However, a Gram-negative bacterium, Sphingomonas sp. A1, has a smart biosystem for the import and depolymerization of macromolecules. The bacterial cells directly incorporate a macromolecule, alginate, into the cytoplasm through a "superchannel", as we named it. The superchannel consists of a pit on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Cytoplasmic polysaccharide lyases depolymerize alginate into the constituent monosaccharides. Other than the proteins characterized so far, novel proteins (e.g., flagellin homologs) have been found to be crucial for the import and depolymerization of alginate through genomics- and proteomics-based identification, thus indicating that the biosystem is precisely constructed and regulated by diverse proteins. In this review, we focus on the structure and function of the bacterial biosystem together with the evolution of related proteins.

Bacterial Proteins↗
PubMed2004

Molecular identification and characterization of an alginate-binding protein on the cell surface of Sphingomonas sp. A1.

Cells of Sphingomonas sp. A1 (strain A1) directly incorporate a macromolecule, alginate, into cytoplasm through a biosystem, or "super-channel," consisting of a pit on the cell surface, alginate-binding proteins in periplasm, and an ABC transporter in the inner membrane. The pit functions as a concentrator for extracellular alginate. Through differential display analysis, a protein (p8) with a molecular mass of 20kDa and a pI of 7.4 was found to be inducibly expressed in the outer membrane of alginate-grown cells. The gene coding for p8 was identified in the genome of strain A1 and shown to be similar to that for the polyhydroxyalkanoate granule-associated protein of Ralstonia eutropha. The disruptant of p8 gene showed significant growth retardation in the alginate medium. An overexpression system for p8 was constructed in Escherichia coli, and the protein was purified and characterized. Surface plasmon resonance biosensor analysis indicated that p8 is able to bind alginate most efficiently at pH 4.0. The above results indicate that p8 is a cell surface protein able to bind alginate and facilitates the concentration of alginate in the pit on the cell surface of strain A1.

Alginates↗
PubMed2004

Practical asymmetric synthesis of beta-hydroxy-beta-trifluoromethylated ketones via the first example of the in situ generation of trifluoro-acetaldehyde and its successive asymmetric carbon-carbon bond formation reaction with chiral imines.

Not only trifluoroactaldehyde ethyl hemiacetal or hydrate but also other polyfluoroalkylaldehydes acetals or hydrates react with an equimolar amount of various chiral imines, followed by hydrolysis to produce the corresponding (S)-beta-hydroxy-beta-polyfluoroalkyl ketones in good yields with good enantioselectivities; furthermore, the ee of the products can be improved by simple recrystallization.

Journal Article↗
PubMed2004

Crystal structure of unsaturated glucuronyl hydrolase, responsible for the degradation of glycosaminoglycan, from Bacillus sp. GL1 at 1.8 A resolution.

Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated d-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 A resolution with a final R-factor of 16.8% for 25 to 1.8 A resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an alpha(6)/alpha(6)-barrel, whose structure is found in the six-hairpin enzyme superfamily of an alpha/alpha-toroidal fold. One side of the UGL alpha(6)/alpha(6)-barrel structure consists of long loops containing three short beta-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Asp(88) and Asp(149). This was supported by site-directed mutagenesis studies in Asp(88) and Asp(149).

Amino Acid Sequence↗
PubMed2004

Structure and function of a hypothetical Pseudomonas aeruginosa protein PA1167 classified into family PL-7: a novel alginate lyase with a beta-sandwich fold.

Structural and functional analyses of alginate lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease. Most alginate lyases are classified into polysaccharide lyase (PL) family-5 and -7 based on their primary structures. Family PL-7 enzymes are still poorly characterized especially in structural properties. Among family PL-7, a gene coding for a hypothetical protein (PA1167) homologous to Sphingomonas alginate lyase A1-II was found to be present in the P. aeruginosa genome. PA1167 overexpressed in Escherichia coli cleaved glycosidic bonds in alginate and released unsaturated saccharides, indicating that PA1167 is an alginate lyase catalyzing a beta-elimination reaction. The enzyme acted preferably on heteropolymeric regions endolytically and worked most efficiently at pH 8.5 and 40 degrees C. The specific activity of PA1167, however, was much weaker than that of the known alginate lyase AlgL, suggesting that AlgL plays a main role in alginate depolymerization in P. aeruginosa. In addition to this specific activity, differences were found between PA1167 and AlgL in enzyme properties such as molecular mass, optimum pH, salt effect, and substrate specificity. The first crystal structure of the family PL-7 alginate lyase was determined at 2.0 A resolution. PA1167 was found to form a glove-like beta-sandwich composed of 15 beta-strands and 3 alpha-helices. The structural difference between the beta-sandwich PA1167 of family PL-7 and alpha/alpha-barrel AlgL of family PL-5 may be responsible for the enzyme characteristics. Crystal structures of polysaccharide lyases determined so far indicate that they can be assigned to three folding groups having parallel beta-helix, alpha/alpha-barrel, and alpha/alpha-barrel + antiparallel beta-sheet structures as basic frames. PA1167 is the fourth novel folding structure found among polysaccharide lyases.

Amino Acid Sequence↗
PubMed2004

Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp. GL1.

Cells of Bacillus sp. GL1 extracellularly secrete a gellan lyase with a molecular mass of 130 kDa responsible for the depolymerization of a heteropolysaccharide (gellan), although the gene is capable of encoding a huge protein with a molecular mass of 263 kDa. A maturation route for gellan lyase in the bacterium was determined using anti-gellan lyase antibodies. The fluid of the bacterial exponentially growing cultures on gellan contained two proteins with molecular masses of 260 and 130 kDa, both of which reacted with the antibodies. The 260 kDa protein was purified from the cultured fluid and characterized. The protein exhibited gellan lyase activity and showed similar enzyme properties, such as optimal pH and temperature, thermal stability, and substrate specificity, to those of the 130 kDa gellan lyase. The N-terminal amino acid sequences of the 260 and 130 kDa enzymes were found to be identical. Determination of the C-terminal amino acid of the 130 kDa enzyme indicated that the 260 kDa enzyme is cleaved between the 1205Gly and 1206Leu residues to yield the mature form (130 kDa) of the gellan lyase. Therefore, the mature enzyme consists of 1170 amino acids (36Ala-1205Gly) with a molecular weight of 125,345, which is in good agreement with that calculated from SDS-PAGE analysis. Judging from these results, gellan lyase is first synthesized as a preproform (263 kDa) and then secreted as a precursor (260 kDa) into the medium through cleavage of the signal peptide. Finally, the precursor is post-translationally processed into the N-terminal half domain of 130 kDa as the mature form, the function of C-terminal half domain being unclear.

Amino Acid Sequence↗
PubMed2004

Super-channel in bacteria: structural and functional aspects of a novel biosystem for the import and depolymerization of macromolecules.

Cells of Sphingomonas sp. A1 directly incorporate a macromolecule, alginate, into the cytoplasm through a biosystem, super-channel, consisting of a pit on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. The alginate is finally depolymerized into constituent monosaccharides by polysaccharide lyases present in the cytoplasm. The fundamental frame of the biosystem for alginate transport, and the functions of the pit, binding proteins, and ABC transporter have already been reviewed together with those of alginate-depolymerization processes [Hashimoto et al., J. Biosci. Bioeng., 87, 123-136 (1999)]. In this review, we have attempted to demonstrate the three-dimensional structure and evolution features of the super-channel, and alginate-depolymerization processes by using information obtained mainly through genomics, proteomics, and X-ray crystallography.

Journal Article↗
PubMed2004

Origin and diversity of alginate lyases of families PL-5 and -7 in Sphingomonas sp. strain A1.

Sphingomonas sp. strain A1 has three endotype alginate lyases (A1-I, A1-II [family PL-7], and A1-III [family PL-5]), each of which is encoded by a single gene. In addition to those of these lyases, a gene (the A1-II' gene) showing significant identity with the A1-II gene was present in the bacterial genome and coded for an alginate lyase with broad substrate specificity. Since no expression of A1-II' was observed even in bacterial cells grown on alginate, the A1-II' gene was thought to be a silent gene derived from the A1-II gene, presumably through duplication, modification, and translocation.

Amino Acid Sequence↗
PubMed2003

Crystallization and preliminary X-ray analysis of alginate lyase, a member of family PL-7, from Pseudomonas aeruginosa.

Alginate lyase depolymerizes alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. A protein PA1167 with a molecular mass of 25 kDa produced by Pseudomonas aeruginosa is an alginate lyase classified into polysaccharide lyase family PL-7. The enzyme was crystallized at 293 K in a drop solution comprising 1.4 M sodium chloride, 0.1 M potassium sodium phosphate and 0.1 M 2-morpholinoethanesulfonate-sodium hydroxide pH 6.5 by means of the vapor-diffusion method. The crystals were monoclinic and belonged to space group P2(1), with unit-cell parameters a = 43.4, b = 70.3, c = 67.4 A, beta = 94.5 degrees. Diffraction data were collected to 2.0 A from a single crystal.

Carbohydrate Sequence↗
PubMed2003

Molecular identification of an alpha-L-rhamnosidase from Bacillus sp strain GL1 as an enzyme involved in complete metabolism of gellan.

The genes (rhaA and rhaB) for two alpha-L-rhamnosidases of Bacillus sp. strain GL1, which assimilates a bacterial polysaccharide (gellan), were cloned from a genomic DNA library of the bacterium constructed in Escherichia coli, and the nucleotide sequences of the genes were determined. Gene rhaA (2661 bp) contained an open reading frame (ORF) encoding a protein (RhaA: 886 amino acids) with a molecular weight (MW) of 98280. Gene rhaB (2871 bp) contained an ORF encoding a protein (RhaB: 956 amino acids) with a MW of 106049. RhaA exhibited significant identity (41%) with alpha-L-rhamnosidase of Clostridium stercorarium, while RhaB showed slight homology with enzymes from other sources. An overexpression system for the two enzymes was constructed in E. coli, and the enzymes were purified and characterized. Both RhaA and RhaB were highly specific for rhamnosyl saccharides, including gellan disaccharide (rhamnosyl glucose) and naringin, and released rhamnose from substrates most efficiently at pH 6.5-7.0 and 40 degrees C. Bacillus sp. strain GL1 cells grown in a gellan medium produced only RhaB, indicating that RhaB plays a crucial role in the complete metabolism of gellan.

Amino Acid Sequence↗