Biomedical subjects

Kousaku Murata

Publications and source records attributed to Kousaku Murata.

At least 19 recordsLinked to original sources

PubMed2007

A structural factor responsible for substrate recognition by Bacillus sp. GL1 xanthan lyase that acts specifically on pyruvated side chains of xanthan.

Xanthan is a bacterial heteropolysaccharide composed of pentasaccharide repeating units, i.e., a cellobiose as a backbone and a trisaccharide consisting of two mannoses and one glucuronic acid as a side chain. Nonreducing terminal mannose residues of xanthan side chains are partially pyruvated. Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8, acts specifically on pyruvated side chains of xanthan and yields pyruvated mannose through a beta-elimination reaction by using a single Tyr255 residue as base and acid catalysts. Here we show structural factors for substrate recognition by xanthan lyase through X-ray crystallographic and mutational analyses. The enzyme accommodates mannose and pyruvated mannose at the -1 subsite, although both inhibitor and dissociation constants of the two monosaccharides indicated that the affinity of pyruvated mannose for xanthan lyase is much higher than that of mannose. The high affinity of pyruvated mannose is probably due to the formation of additional hydrogen bonds between the carboxyl group of pyruvated mannose and amino acid residues of Tyr315 and Arg612. Site-directed mutagenesis of the two residues demonstrated that Arg612 is a key residue in recognizing pyruvated mannose. Arg612 is located in the protruding loop covering the substrate, suggesting that the loop functions as a lid that is responsible for the proper accommodation of the substrate at the active site.

Bacillus↗

PubMed2006

Crystal structure of unsaturated glucuronyl hydrolase complexed with substrate: molecular insights into its catalytic reaction mechanism.

Unsaturated glucuronyl hydrolase (UGL), which is a member of glycoside hydrolase family GH-88, is a bacterial enzyme that degrades mammalian glycosaminoglycans and bacterial biofilms. The enzyme, which acts on unsaturated oligosaccharides with an alpha-glycoside bond produced by microbial polysaccharide lyases responsible for bacterial invasion of host cells, was believed to release 4-deoxy-l-threo-5-hexosulose-uronate (unsaturated glucuronic acid, or DeltaGlcA) and saccharide with a new nonreducing terminus by hydrolyzing the glycosidic bond. We detail the crystal structures of wild-type inactive mutant UGL of Bacillus sp. GL1 and its complex with a substrate (unsaturated chondroitin disaccharide), identify active site residues, and postulate a reaction mechanism catalyzed by UGL that triggers the hydration of the vinyl ether group in DeltaGlcA, based on the structural analysis of the enzyme-substrate complex and biochemical analysis. The proposed catalytic mechanism of UGL is a novel case among known glycosidases. Under the proposed mechanism, Asp-149 acts as a general acid and base catalyst to protonate the DeltaGlcA C4 atom and to deprotonate the water molecule. The deprotonated water molecule attacks the DeltaGlcA C5 atom to yield unstable hemiketal; this is followed by spontaneous conversion to an aldehyde (4-deoxy-l-threo-5-hexosulose-uronate) and saccharide through hemiacetal formation and cleavage of the glycosidic bond. UGL is the first clarified alpha(6)/alpha(6)-barrel enzyme using aspartic acid as the general acid/base catalyst.

Bacillus↗

PubMed2006

Structure of unsaturated rhamnogalacturonyl hydrolase complexed with substrate.

Bacillus subtilis strain 168 YteR has been identified as a novel enzyme "unsaturated rhamnogalacturonyl hydrolase" classified in glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) produced from plant cell wall RG type-I treated with RG lyases, releasing unsaturated galacturonic acid (DeltaGalA) from the substrate. The most likely candidate catalytic residue is Asp-143. Here, we show the structure of D143N in complex with unsaturated RG disaccharide (substrate) determined at 1.9A resolution by X-ray crystallography. This structural feature directly contributes to the postulation of the enzyme reaction mechanism. YteR triggers the hydration of vinyl ether group in DeltaGalA, but not of glycoside bond, by using Asp-143 as a general acid and base catalyst. Asp-143 donates proton to the double bond of DeltaGalA as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of DeltaGalA.

Amino Acid Sequence↗

PubMed2006

Paenibacillus sp. strain HC1 xylanases responsible for degradation of rice bran hemicellulose.

Paenibacillus sp. strain HC1 is the first bacterium capable of growing on rice bran hemicellulose as a sole carbon source. Two xylanases (Xyl-I and -II) were purified from the bacterial culture fluid and enzymatically characterized. Xyl-I and -II showed monomer forms with molecular masses of 30 and 18kDa, respectively, and were most active at around pH 5.0 and 45 degrees C. Xylooligosaccharides were degraded to xylobiose and xylose by Xyl-I, but not by Xyl-II, suggesting that Xyl-I plays an important role in complete depolymerization of xylan. Both enzymes acted endolytically on rice bran hemicellulose, indicating that Xyl-I and -II contribute to the structure determination and practical use of the polysaccharide, an unutilized biomass in technology.

Dietary Fiber↗

PubMed2006

Crystallization and preliminary crystallographic analysis of the family GH78 alpha-L-rhamnosidase RhaB from Bacillus sp. GL1.

Alpha-L-rhamnosidases play important roles in the metabolism of plant cell walls, glycosides and bacterial biofilms. This enzyme is also used industrially for debittering citrus fruits by releasing rhamnose from the plant flavonoid naringin. Bacillus sp. GL1 alpha-L-rhamnosidase (RhaB) is a member of glycoside hydrolase (GH) family 78. Native and selenomethionine-derivative enzymes were crystallized at 293 K by hanging-drop vapour diffusion with polyethylene glycol 8000 as a precipitant. This is the first report of the crystallization of a family GH78 enzyme.

Bacillus↗

PubMed2006

Substrate recognition by unsaturated glucuronyl hydrolase from Bacillus sp. GL1.

Bacterial unsaturated glucuronyl hydrolases (UGLs) together with polysaccharide lyases are responsible for the complete depolymerization of mammalian extracellular matrix glycosaminoglycans. UGL acts on various oligosaccharides containing unsaturated glucuronic acid (DeltaGlcA) at the nonreducing terminus and releases DeltaGlcA through hydrolysis. In this study, we demonstrate the substrate recognition mechanism of the UGL of Bacillus sp. GL1 by determining the X-ray crystallographic structure of its substrate-enzyme complexes. The tetrasaccharide-enzyme complex demonstrated that at least four subsites are present in the active pocket. Although several amino acid residues are crucial for substrate binding, the enzyme strongly recognizes DeltaGlcA at subsite -1 through the formation of hydrogen bonds and stacking interactions, and prefers N-acetyl-d-galactosamine and glucose rather than N-acetyl-d-glucosamine as a residue accommodated in subsite +1, due to the steric hindrance.

Acetylgalactosamine↗

PubMed2006

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide.

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.

Acetylglucosamine↗

PubMed2006

Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15.

Almost all alginate lyases depolymerize alginate in an endolytical fashion via a beta-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2(1) and diffracted to 2.8 angstroms resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 angstroms, beta = 91.5 degrees.

Agrobacterium tumefaciens↗

PubMed2006

Crystallization and preliminary X-ray analysis of the rhamnogalacturonan lyase YesW from Bacillus subtilis strain 168, a member of polysaccharide lyase family 11.

Rhamnogalacturonan lyases degrade rhamnogalacturonan I, a major component of pectin, through a beta-elimination reaction. YesW from Bacillus subtilis strain 168 is a novel rhamnogalacturonan lyase classified into polysaccharide lyase family 11 (PL-11). The enzyme was crystallized at 293 K using the sitting-drop vapour-diffusion method with 2-methyl-2,4-pentanediol (MPD) as a precipitant. Preliminary X-ray analysis revealed that the YesW crystals belong to space group P2(1) and diffract to 2.40 angstroms resolution, with unit-cell parameters a = 56.7, b = 105.6, c = 101.4 A, beta = 94.9 degrees. This is the first report on the crystallization and preliminary X-ray analysis of a family PL-11 rhamnogalacturonan lyase.

Bacillus subtilis↗

PubMed2006

Overexpression, purification and preliminary X-ray analysis of pullulanase from Bacillus subtilis strain 168.

The AmyX gene encoding pullulanase from the common spore-forming bacterium Bacillus subtilis strain 168 was cloned, overexpressed in Escherichia coli, purified and crystallized. The recombinant pullulanase was purified to homogeneity using ammonium sulfate precipitation, hydrophobic chromatography and anion-exchange chromatography, resulting in a specific activity of 24.10 U per milligram of protein. SDS-PAGE analysis showed that the molecular weight of the protein is approximately 81.0 kDa, which is similar to the calculated molecular weight, 81.1 kDa, from its translated cDNA sequence. The k(cat) and K(m) of the purified enzyme with pullulan as substrate were approximately 79 s(-1) and 1.284 mg ml(-1), respectively. X-ray crystallographic analysis of the pullulanase crystal showed that the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 70.568, b = 127.68, c = 189.25 angstroms. The crystal contains two molecules of pullulanase in the asymmetric unit, with a solvent content of 53.15%. The crystal diffracted to 2.1 angstroms resolution at a synchrotron and is suitable for structure determination.

Bacillus subtilis↗

PubMed2006

A biosystem for alginate metabolism in Agrobacterium tumefaciens strain C58: molecular identification of Atu3025 as an exotype family PL-15 alginate lyase.

The Gram-negative bacterium Sphingomonas sp. strain A1 (strain A1) has a peculiar biosystem to directly import and depolymerize a macromolecule, alginate, which is encoded by a cluster of genes on the genome. We identified five clustered ORFs homologous to some genes of the strain A1 cluster in the genome of Agrobacterium tumefaciens strain C58 (strain C58). These ORFs are Atu3021, Atu3022, Atu3023, and Atu3024, encoding a putative sugar ABC transporter system and Atu3025, which encodes a putative alginate lyase. We analyzed the involvement of this gene cluster in alginate metabolism. Strain C58 cells grew significantly on low-molecular-weight (LMW) alginate (average molecular weight, 1000), and we detected specific alginate-induced expression of Atu3024 and Atu3025. This strain does not grow on alginate (average molecular weight, 25,600), suggesting that the strain C58 gene cluster is involved in importing and degrading LMW alginate. One protein, Atu3025, purified from strain C58, was identified as an alginate lyase, and the enzyme overexpressed in Escherichia coli was further characterized. Atu3025 released monosaccharides specifically from alginate most efficiently at pH 7.3 and 30 degrees C through a beta-elimination reaction, indicating that Atu3025 is an exotype alginate lyase potentially involved in the assimilation of LMW alginate in strain C58.

Agrobacterium tumefaciens↗

PubMed2006

Sequence and analysis of the 46.6-kb plasmid pA1 from Sphingomonas sp. A1 that corresponds to the typical IncP-1beta plasmid backbone without any accessory gene.

Sphingomonas sp. A1 (strain A1) is capable of directly incorporating macromolecules (e.g., alginate) through the specialized import system--"super-channel." Here, we report the complete DNA sequence and genetic organization of plasmid pA1 from strain A1. Nucleotide sequence analysis revealed that pA1 comprises 46,557 bp encoding 49 open reading frames (ORFs) with 65% G+C content and abundant GCCG/CGGC motifs. Many predicted pA1 ORFs showed high similarity to pA81 ORFs; pA81 is supposedly a self-transmissible promiscuous incompatibility (Inc) group P-1beta plasmid. Unlike any reported IncP-1 plasmids, pA1 contains no inserted mobile genetic elements. The genetic organization and predicted pA1 ORFs showed greater similarity to the IncP-1beta plasmid backbone than to the IncP-1alpha plasmid backbone. pA1 contains restriction site-associated repeat sequences typical of the IncP-1beta but absent in the IncP-1alpha and delta subgroups. Thus, the overall pA1 structure corresponds to that of the typical IncP-1beta plasmids. Phylogenetic analysis of the replication-associated proteins suggested that pA1 may have diverged later along with the two IncP-1beta plasmids--pA81 and pB4. The 2.4-kb duplicates of stable inheritance genes klcAB and korC in pA1 possibly resulted from insertion and/or recombination events via the repeat sequences flanking these duplicates.

Base Sequence↗

PubMed2006

Engineered membrane superchannel improves bioremediation potential of dioxin-degrading bacteria.

Sphingomonas sp. A1 possesses specialized membrane structures termed 'superchannels' that enable the direct incorporation of macromolecules into the cell. We have engineered two related sphingomonads, the dioxin-degrading S. wittichii RW1 and the polypropylene glycol-degrading S. subarctica IFO 16058(T), to incorporate this superchannel into their cell membranes. In both cases the bioremediation capability of the organisms was substantially increased pointing at the potential of this approach as a general strategy to improve bacterial degradation of hazardous compounds in the environment.

Biodegradation, Environmental↗

PubMed2005

Degradation of rice bran hemicellulose by Paenibacillus sp. strain HC1: gene cloning, characterization and function of beta-D-glucosidase as an enzyme involved in degradation.

A bacterium (strain HC1) capable of assimilating rice bran hemicellulose was isolated from a soil and identified as belonging to the genus Paenibacillus through taxonomical and 16S rDNA sequence analysis. Strain HC1 cells grown on rice bran hemicellulose as a sole carbon source inducibly produced extracellular xylanase and intracellular glycosidases such as beta-D-glucosidase and beta-D-arabinosidase. One of them, beta-D-glucosidase, was further analyzed. A genomic DNA library of the bacterium was constructed in Escherichia coli and gene coding for beta-D-glucosidase was cloned by screening for beta-D-glucoside-degrading phenotype in E. coli cells. Nucleotide sequence determination indicated that the gene for the enzyme contained an open reading frame consisting of 1,347 bp coding for a polypeptide with a molecular mass of 51.4 kDa. The polypeptide exhibits significant homology with other bacterial beta-D-glucosidases and belongs to glycoside hydrolase family 1. Beta-D-Glucosidase purified from E. coli cells was a monomeric enzyme with a molecular mass of 50 kDa most active at around pH 7.0 and 37 degrees C. Strain HC1 glycosidases responsible for degradation of rice bran hemicellulose are expected to be useful for structurally determining and molecularly modifying rice bran hemicellulose and its derivatives.

Amino Acid Sequence↗

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

MJ0917 in archaeon Methanococcus jannaschii is a novel NADP phosphatase/NAD kinase.

NAD kinase phosphorylates NAD(+) to form NADP(+). Conversely, NADP phosphatase, which has not yet been identified, dephosphorylates NADP(+) to produce NAD(+). Among the NAD kinase homologs, the primary structure of MJ0917 of hyperthermophilic archaeal Methanococcus jannaschii is unique. MJ0917 possesses an NAD kinase homologous region in its C-terminal half and an inositol-1-phosphatase homologous region in its N-terminal half. In this study, MJ0917 was biochemically shown to possess both NAD kinase and phosphatase activities toward NADP(+), NADPH, and fructose 1,6-bisphosphate, but not toward inositol 1-phosphate. With regard to the phosphatase activity, kinetic values indicated that NADP(+) is the preferred substrate and that MJ0917 would function as a novel NADP phosphatase/NAD kinase showing conflicting dual activities, viz. synthesis and degradation of an essential NADP(+). Furthermore, in vitro analysis of MJ0917 showed that, although MJ0917 could supply NADP(+), it prevented excess accumulation of NADP(+); thus, it has the ability to maintain a high NAD(+)/NADP(+) ratio, whereas 5'-AMP would decrease this ratio. The evolutionary process during which MJ0917 arose is also discussed.

Archaeal Proteins↗

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↗