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S Kuramitsu

Publications and source records attributed to S Kuramitsu.

At least 37 records · Page 2Linked to original sources
PubMed2000

The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii.

Aromatic amino acid aminotransferase (ArATPh), which has a melting temperature of 120 degrees C, is one of the most thermostable aminotransferases yet to be discovered. The crystal structure of this aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii was determined to a resolution of 2.1 A. ArATPh has a homodimer structure in which each subunit is composed of two domains, in a manner similar to other well characterized aminotransferases. By the least square fit after superposing on a mesophilic ArAT, the ArATPh molecule exhibits a large deviation of the main chain coordinates, three shortened alpha-helices, an elongated loop connecting two domains, and a long loop transformed from an alpha-helix, which are all factors that are likely to contribute to its hyperthermostability. The pyridine ring of the cofactor pyridoxal 5'-phosphate covalently binding to Lys(233) is stacked parallel to F121 on one side and interacts with the geminal dimethyl-CH/pi groups of Val(201) on the other side. This tight stacking against the pyridine ring probably contributes to the hyperthermostability of ArATPh. Compared with other ArATs, ArATPh has a novel substrate specificity, the order of preference being Tyr > Phe > Glu > Trp > His>> Met > Leu > Asp > Asn. Its relatively weak activity against Asp is due to lack of an arginine residue corresponding to Arg(292)* (where the asterisk indicates that this is a residues supplied by the other subunit of the dimer) in pig cytosolic aspartate aminotransferase. The enzyme recognizes the aromatic substrate by hydrophobic interaction with aromatic rings (Phe(121) and Tyr(59)*) and probably recognizes acidic substrates by a hydrophilic interaction involving a hydrogen bond network with Thr(264)*.

Amino Acid Sequence↗
PubMed2000

Structural genomics projects in Japan.

Two major structural genomics projects exist in Japan. The oldest, the RIKEN Structural Genomics Initiative, has two major goals: to determine bacterial, mammalian, and plant protein structures by X-ray crystallography and NMR spectroscopy and to perform functional analyses with the target proteins. The newest, the structural genomics project at the Biological Information Research Center, focuses on human membrane proteins.

Animals↗
PubMed2000

The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 degrees C.

The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 degrees C. It also showed high stability of 18.3 kcal.mol-1 (76.5 kJ.mol-1) at 25 degrees C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecA-like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange. At 75 degrees C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 degrees C. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 degrees C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA-dependent ATPase activity of P. islandicum RadA displays a temperature-dependent capacity to exist in two different catalytic modes, with 75 degrees C being the critical threshold temperature.

Adenosine Triphosphatases↗
PubMed2000

Identification of Thermus thermophilus HB8 tRNA (Gm18) methyltransferase gene.

For the purpose of identification of the gene for Thermus thermophilus tRNA (Gm18) methyltransferase [tRNA (guanosine-2'-)-methyltransferase, EC 2.1.1.34], the purified enzyme from native source was analyzed by the peptide-mass mapping. The target gene encoded the amino acid sequences of the obtained peptides was searched in data from Thermus thermophilus HB8 genome-sequencing project. We found the target gene AB05130, which was expected to encode a protein composed of 194 amino acid residues and the molecular mass of this protein was calculated as 22083. The recombinant protein was expressed in E. coli as an active form. The Gm18 formation activity of the purified recombinant protein was confirmed by in vitro methylation followed by two-dimensional thin layer chromatography and Liquid Chromatography/Mass Spectrum analysis of substrate tRNA.

Base Sequence↗
PubMed2000

Crystallization and preliminary X-ray crystallographic studies of Thermus thermophilus HB8 MutM protein involved in repairs of oxidative DNA damage.

MutM protein, which removes the oxidatively damaged DNA base product, 8-oxoguanine (GO), has been crystallized by means of a hanging-drop vapor-diffusion procedure using polyethyleneglycol monomethylether 2000 as a precipitant in 2-(cyclohexylamino) ethanesulfonic acid (CHES) buffer, pH 9.8. The diffraction data derived from oscillation photographs indicate that the crystals belong to the monoclinic system and space group P2(1). The crystals have unit-cell dimensions of a = 45.4 A, b = 62.0 A, c = 99.7 A, and beta = 90.8 degrees. Assuming that the asymmetric unit contains two molecules, the Vm value was calculated to be 2.35 A(3).Da(-1). The crystals diffracted X-rays to at least 2.1 A resolution and were suitable for high-resolution X-ray crystal structure determination.

Crystallization↗
PubMed2000

Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).

Adenosine Triphosphate↗
PubMed2000

Identification and characterization of Thermus thermophilus HB8 RuvA protein, the subunit of the RuvAB protein complex that promotes branch migration of Holliday junctions.

The Escherichia coli ruvA and ruvB genes constitute an SOS-regulated operon. The products of these genes form a protein complex that promotes branch migration of the Holliday junction, an intermediate of homologous recombination. RuvA protein binds specifically to the Holliday junction and recruits RuvB protein to the junction. RuvB is an ATP-driven motor protein involved in branch migration. We previously cloned the ruvB gene of the thermophilic bacterium Thermus thermophilus HB8 (Tth) and found that, in contrast to the operon structure in most mesothermic bacteria, the ruvA gene is absent from the vicinity of ruvB. In this work, we cloned the ruvA gene from T. thermophilus HB8 and analyzed its nucleotide sequence. Tth RuvA is a protein of 20,414 Da consisting of 191 amino acid residues, and is 37% identical in amino acid sequence to E. coli RuvA. Tth ruvA complemented the DNA repair defect of E. coli deltaruvA mutants. The purified Tth RuvA protein stimulated Tth RuvB activities, such as hydrolysis of ATP and promotion of branch migration of the Holliday junction, in a manner similar to the RuvA-RuvB interactions observed in E. coli. In addition, Tth RuvA stimulated the E. coli RuvB activities in vitro, which was well consistent with the results of in vivo hetero-complementation experiments.

Adenosine Triphosphatases↗
PubMed1999

Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.

Amino Acid Substitution↗
PubMed1999

Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo-keto reductase superfamily.

Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP(+), was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 +/- 1,600. The enzyme showed a single absorption peak at 280 nm (E(1%) = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank(TM) accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K(+) channel beta2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.

Alcohol Oxidoreductases↗
PubMed1999

Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate.

The three-dimensional structures of pyridoxal 5'-phosphate-type aspartate aminotransferase (AspAT) from Thermus thermophilus HB8 and pyridoxamine 5'-phosphate type one in complex with maleate have been determined by X-ray crystallography at 1.8 and 2.6 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. AspATs from many species were classified into aminotransferase subgroups Ia and Ib. The enzyme belongs to subgroup Ib, its sequence being less than 16% identical to the primary sequences of Escherichia coli, pig cytosolic, and chicken mitochondrial AspATs, which belong to subgroup Ia whose sequences are more than 40% identical and whose three-dimensional structures are quite similar with the active site residues almost completely conserved. The first X-ray analysis of AspAT subgroup Ib indicated that the overall and the active site structures are essentially conserved between the AspATs of subgroup Ia and the enzyme of subgroup Ib, but there are two distinct differences between them. (1) In AspAT subgroup Ia, substrate (or inhibitor) binding induces a large movement of the small domain as a whole to close the active site. However, in the enzyme of subgroup Ib, only the N-terminal region (Lys13-Val30) of the small domain approaches the active site to interact with the maleate. (2) In AspAT subgroup Ia, Arg292 recognizes the side chain carboxylate of the substrate; however, residue 292 of the enzyme in subgroup Ib is not Arg, and in place of Arg292, Lys109 forms a salt bridge with the side chain carboxylate. The thermostability of the enzyme is attained at least in part by the high content of Pro residues in the beta-turns and the marked increase in the number of salt bridges on the molecular surface compared with the mesophilic AspAT.

Amino Acid Sequence↗
PubMed1999

Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA.

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.

Adenosine Triphosphatases↗
PubMed1999

Cloning of the RNA polymerase alpha subunit gene from Thermus thermophilus HB8 and characterization of the protein.

The region containing the RNA polymerase alpha subunit (RNAPalpha) gene (rpoA) and the ribosomal protein genes of a thermophilic eubacterial strain, Thermus thermophilus (Tt) HB8, was cloned from a genomic DNA library by Southern hybridization. The gene order in this region is rpl36-rps13-rps11-rps4-rpoA-rpl17, which is identical to that in some other eubacteria. The rpoA gene encodes a 315 amino acid residue protein with a molecular weight of 35,013, the amino acid sequence showing 42% identity to that of Escherichia coli (Ec). From the results of comparison of the amino acid sequence and the predicted secondary structure of the C-terminal domain of Tt RNAPalpha (Tt alphaCTD) with those of Ec, the overall folding is expected to be similar. However, amino acid residues Asn268 and Cys269 in Ec alphaCTD, which are essential for its interaction with DNA or regulatory proteins, were replaced by His and Ser, respectively, in Tt alphaCTD. By means of a T7-based expression system in Ec cells, Tt RNAPalpha was overexpressed and purified. The high thermostability of Tt RNAPalpha was demonstrated by the CD spectra.

Amino Acid Sequence↗
PubMed1999

Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus.

The whole-genome sequencing of an extreme thermophile, Thermus thermophilus, is now in progress. Like other genome projects, major concern is shifting from the sequence itself to post-sequencing research such as functional or structural genomics. Under such circumstances, the demand for convenient genetic-engineering tools is increasing. In this study we have increased the thermostability of a kanamycin-resistance gene product using strategies based on directed evolution in T. thermophilus to the upper limit of its growth temperature. The most thermostable mutant has 19 amino-acid substitutions, whereby the thermostability is increased by 20 degrees C, but the enzymatic activity is not significantly changed. Most of the mutated residues are located on the surface of the protein molecule, and, interestingly, five of the 19 substitutions are those to proline residues. The evolved kanamycin-resistance gene products could be used as selection markers at the optimum growth temperature of T. thermophilus. The development of such a convenient genetic-engineering tool would facilitate post-sequencing research on T. thermophilus.

Amino Acid Substitution↗
PubMed1999

Crystal structure of Thermus thermophilus HB8 UvrB protein, a key enzyme of nucleotide excision repair.

In the nucleotide excision repair system, UvrB plays a central role in damage recognition and DNA incision by interacting with UvrA and UvrC. We have determined the crystal structure of Thermus thermophilus HB8 UvrB at 1.9 A resolution. UvrB comprises four domains, two of which have an alpha/beta structure resembling the core domains of DNA and RNA helicases. Additionally, UvrB has an alpha-helical domain and a domain consisting of antiparallel beta-sheets (beta-domain). The sequence similarity suggests that the beta-domain interacts with UvrA. Based on the distribution of the conserved regions and the structure of the PcrA-DNA complex, a model for the UvrB-DNA complex is proposed.

Bacterial Proteins↗
PubMed1999

Crystallization and preliminary X-ray diffraction studies of a DNA excision repair enzyme, UvrB, from Thermus thermophilus HB8.

A DNA excision repair enzyme, UvrB, from Thermus thermophilus HB8 was crystallized by the vapor-diffusion method using lithium sulfate as the precipitant and beta-octylglucoside as an additive. The crystals belong to the trigonal space group P3121 or P3221, with unit-cell dimensions of a = b = 136.0 and c = 108.1 A. The crystal is most likely to contain one UvrB protein in an asymmetric unit with the Vm value of 3.8 A3 Da-1. The crystals diffracted X-rays beyond 2.9 A resolution. Although the crystals were sensitive to X-ray irradiation at room temperature, the frozen crystals at 100 K showed no apparent decay during the intensity measurement.

Bacterial Proteins↗
PubMed1998

The novel substrate recognition mechanism utilized by aspartate aminotransferase of the extreme thermophile Thermus thermophilus HB8.

Aspartate aminotransferase (AspAT) is a unique enzyme that can react with two types of substrate with quite different properties, acidic substrates, such as aspartate and glutamate, and neutral substrates, although the catalytic group Lys-258 acts on both types of substrate. The dynamic properties of the substrate-binding site are indispensable to the interaction with hydrophobic substrates (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem. (Tokyo) 122, 55-63). AspATs from various organisms are classified into two subgroups, Ia and Ib. The former includes AspATs from Escherichia coli and higher eukaryotes, whereas the latter includes those from Thermus thermophilus and many prokaryotes. The AspATs belonging to subgroup Ia each have an Arg-292 residue, which interacts with the distal carboxyl groups of dicarboxylic (acidic) substrates, but the functionally similar residue of subgroup Ib AspATs has not been identified. In view of the x-ray crystallographic structure of T. thermophilus AspAT, we expected Lys-109 to be this residue in the subgroup Ib AspATs and constructed K109V and K109S mutants. Replacing Lys-109 with Val or Ser resulted in loss of activity toward acidic substrates but increased that toward the neutral substrate, alanine, considerably. These results indicate that Lys-109 is a major determinant of the acidic substrate specificity of subgroup Ib AspATs. Kinetic analysis of the interactions with neutral substrates indicated that T. thermophilus AspAT is subject to less steric hindrance and its substrate-binding pocket has a more flexible conformation than E. coli AspAT. A flexible active site in the rigid T. thermophilus AspAT molecule may explain its high activity even at room temperature.

Amino Acid Sequence↗