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Isao Tanaka

Publications and source records attributed to Isao Tanaka.

At least 55 records · Page 3Linked to original sources
PubMed2005

Elucidation of stability determinants of cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Colwellia maris, by construction of chimeric enzymes.

To elucidate determinants of differences in thermostability between mesophilic and psychrophilic monomeric isocitrate dehydrogenases (IDHs) from Azotobacter vinelandii (AvIDH) and Colwellia maris (CmIDH), respectively, chimeric enzymes derived from the two IDHs were constructed based on the recently resolved three-dimensional structure of AvIDH, and several characteristics of the two wild-type and six chimeric IDHs were examined. These characteristics were then compared with those of dimeric IDH from Escherichia coli (EcIDH). All recombinant enzymes with a (His)(6)-tag attached to the N-terminal were overexpressed in the E. coli cells and purified by Ni(2+)-affinity chromatography. The catalytic activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) of the wild-type AvIDH and CmIDH were higher than those of EcIDH, implying that an improved catalytic rate more than compensates for the loss of a catalytic site in the former two IDHs due to monomerization. Analyses of the thermostability and kinetic parameters of the chimeric enzymes indicated that region 2, corresponding to domain II, and particularly region 3 located in the C-terminal part of domain I, are involved in the thermolability of CmIDH, and that the corresponding two regions of AvIDH are important for exhibiting higher catalytic activity and affinity for isocitrate than CmIDH. The relationships between the stability, catalytic activity and structural characteristics of AvIDH and CmIDH are discussed.

Adaptation, Physiological↗
PubMed2005

Preparation, crystallization and preliminary X-ray diffraction analysis of PH1948, predicted RNA methyltransferase from Pyrococcus horikoshii.

RNA methyltransferase is responsible for transferring methyl and resulting in methylation on the bases or ribose ring of RNA, which existed widely but mostly remains an open question. A recombinant protein PH1948 predicting RNA methyltransferase from Pyrococcus horikoshii OT3 has been crystallized. The crystals of selenomethionyl PH1948 belong to space group C2, with unit-cell parameters a=207.0 A, b=43.1 A, c=118.2 A, b=92.1 degrees , and diffract X-rays to 2.2 A resolution. The V(M) value was determined to be 2.8 A3/Da, indicating the presence of four protein molecules in the asymmetric unit.

Crystallization↗
PubMed2005

Increased glucose tolerance in N-type Ca2+ channel alpha(1B)-subunit gene-deficient mice.

The voltage-dependent N-type Ca2+ channel is localized in the plasma membrane of insulin-releasing beta-cells and glucagon-releasing alpha-cells in the islets of Langerhans in the pancreas. To examine the contribution of N-type Ca2+ channel to glucose homeostasis, we performed glucose tolerance and insulin tolerance tests with N-type Ca2+ channel alpha(1B)-subunit-deficient mice on a normal or high-fat diet. The fasting glucose level in homozygous mice on the normal diet was significantly lower than those in wild and heterozygous mice. In glucose tolerance tests, the homozygous mice showed a higher glucose clearance rate and a similar pattern of insulin levels to those of wild and heterozygous mice. In insulin tolerance tests, glucose clearance rates showed no significant difference among wild, heterozygous and homozygous mice. In animals on the high-fat diet, food consumption was the same among wild, heterozygous and homozygous mice, but body weight gain was reduced in homozygous mice. After 8 weeks of the high-fat diet, homozygous mice showed lower fasting glucose levels and exhibited higher glucose clearance and lower insulin levels than wild or heterozygous mice in glucose tolerance tests. Glucose clearance rates showed no significant difference among wild, heterozygous and homozygous mice in insulin tolerance tests. After 10 weeks of the high-fat diet, the alpha(1B)-deficient homozygous mice showed lower lipid deposition in liver and lower plasma glucagon, leptin and triglyceride levels than wild or heterozygous mice. These results suggest that N-type Ca2+ channels play a role in insulin and glucagon release, and that N-type Ca2+ channel alpha(1B)-subunit deficient mice show improved glucose tolerance without any change in insulin sensitivity. Thus, N-type Ca2+ channel blockers might be candidate anti-diabetic/anti-obesity agents.

Animals↗
PubMed2005

Propionibacterium acnes is the most common bacterium commensal in peripheral lung tissue and mediastinal lymph nodes from subjects without sarcoidosis.

BACKGROUND: In studies of the unknown etiology of sarcoidosis, Propionibacterium acnes (a possible agent) was found in the lungs and lymph nodes of many sarcoidosis patients and some control subjects. P. acnes might be commensal not only to the skin, conjunctivae, and intestine, but also to the lungs and lymph nodes of individuals without sarcoidosis. METHODS: We cultured peripheral lung tissue and various lymph nodes obtained from patients with diseases other than sarcoidosis. DNA of 45 isolates of P. acnes from these patients, 67 isolates from normal skin, conjunctiva, and intestine, and 39 isolates from sarcoid lymph nodes were compared by random amplified polymorphic DNA analysis. RESULTS: P. acnes was isolated from half of 43 lungs and 8 of 11 mediastinal lymph nodes, mostly in pure culture. P. acnes was isolated from half of 20 gastric and 3 of 12 intestinal lymph nodes; intestinal bacteria were also numerous. In general, fewer than 500 colony-forming units of P. acnes per gram tissue were isolated, but 4 lung tissue specimens, 2 of which had a few granulomas, had many more. P. acnes strains from a particular site (lung, lymph node, skin or conjunctivae, and intestine) were genetically similar, more than isolates obtained from different sites. Lymph-node isolates from subjects with and without sarcoidosis differed little. CONCLUSION: These results suggest that P. acnes normally resides in peripheral lung tissue and mediastinal lymph nodes and that the strains of P. acnes isolated from sarcoid lymph nodes were not specific to sarcoidosis.

Adult↗
PubMed2004

Crystal structure of the Pyrococcus horikoshii isopropylmalate isomerase small subunit provides insight into the dual substrate specificity of the enzyme.

Recent studies have implied that the isopropylmalate isomerase small subunit of the hyperthermophilic archaea Pyrococcus horikoshii (PhIPMI-s) functions as isopropylmalate isomerase in the leucine biosynthesis pathway, and as homoaconitase (HACN) in the lysine biosynthesis pathway via alpha-aminoadipic acid. PhIPMI is thus considered a key to understanding the fundamental metabolism of the earliest organisms. We describe for the first time the crystal structure of PhIPMI-s, which displays dual substrate specificity. The crystal structure unexpectedly shows that four molecules create an interlocked assembly with intermolecular disulfide linkages having a skewed 222 point-group symmetry. Although the overall fold of the PhIPMI-s monomer is related closely to domain 4 of the aconitase (ACN), one alpha-helix in the ACN structure is replaced by a short loop with relatively high temperature factor values. Because this region is essential for discriminating the structurally similar substrate based on interactions with its diversified gamma-moiety, the loop structure in the PhIPMI-s must be dependent on the presence of a substrate. The flexibility of the loop region might be a structural basis for recognizing both hydrophobic and hydrophilic gamma-moieties of two distinct substrates, isopropylmalate and homocitrate.

Amino Acid Sequence↗
PubMed2004

Structural evidence for guanidine-protein side chain interactions: crystal structure of CutA from Pyrococcus horikoshii in 3 M guanidine hydrochloride.

This study was carried out to investigate the structural perturbation of the protein's local structure by the denaturants under non-denaturing conditions. Crystal structure of CutA from an archaeon Pyrococcus horikosii (PhoCutA), a heavy-metal binding protein, was determined at 1.6-angstroms resolution in the presence of 3 M guanidine HCl (GdnHCl). Native PhoCutA has a large number of short intramolecular hydrogen bonds and salt bridges on the protein surface, of which greater than 90% of hydrogen bonds and all salt bridges were retained in 3 M GdnHCl. Hydrogen bonds that disappeared in the GdnHCl crystal structure were mainly located on the protein surface, especially around the structurally perturbed loop, suggesting interactions between peptide groups and GdnHCl. Only a few GdnH+ ions were observed in the crystal structure, although none at the surface, of the protein. Two GdnH+ ions were observed in the center of the trimeric structure, replacing water molecules, and were hydrogen bonded with Asp84 and Asp86 of each chain. The exterior loop from Tyr39 to Lys44, including Trp40-Trp41, was perturbed structurally. Decreases in temperature factors were observed in beta strand 5 and the N terminus of helix 3. These results suggest the specific bindings of GdnH+ with some acidic residues and the non-specific bindings around Trp residues and peptide groups on the protein surface and that binding of GdnHCl to the native protein is limited, resulting in local structural perturbation.

Archaeal Proteins↗
PubMed2004

Structural and enzymatic properties of 1-aminocyclopropane-1-carboxylate deaminase homologue from Pyrococcus horikoshii.

1-Aminocyclopropane-l-carboxylate deaminase (ACCD) is a pyridoxal 5/-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield a-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity, the GC-MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5'-phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity.

Amino Acid Sequence↗
PubMed2004

Preparation and preliminary X-ray analysis of the catalytic module of beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4.

Beta-1,3-xylanase (1,3-beta-D-xylan xylanohydrolase; EC 3.2.1.32) is an enzyme capable of hydrolyzing beta-1,3-xylan. The newly cloned beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 (XYL4) exhibited a modular structure consisting of three modules: an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two C-terminal xylan-binding modules belonging to carbohydrate-binding module family 31. Despite substantial crystallization screening, crystallization of the recombinant XYL4 was not accomplished. However, the deletion mutant of XYL4, composed of a catalytic module without a xylan-binding module, was crystallized. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 51.6, b = 75.8, c = 82.0 A. X-ray diffraction data were collected to 1.44 A resolution.

Catalytic Domain↗
PubMed2004

Crystallization and preliminary X-ray diffraction study of the catalytic subunit of archaeal H+ -transporting ATP synthase from Pyrococcus horikoshii OT3.

H+ -transporting ATP synthase (H+ -ATPase) is a multi-subunit complex which acts to produce ATP molecules. The catalytic subunit A of the archaeal-type H+ -ATPase from Pyrococcus horikoshii OT3 was cloned, expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method with MPD as a precipitant. X-ray intensity data were collected to 2.55 A resolution at beamline BL41XU of SPring-8. The crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 128.0, c = 104.7 A, and contain one molecule per asymmetric unit.

Catalytic Domain↗
PubMed2004

Crystal structure of the regulatory subunit of archaeal initiation factor 2B (aIF2B) from hyperthermophilic archaeon Pyrococcus horikoshii OT3: a proposed structure of the regulatory subcomplex of eukaryotic IF2B.

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine-nucleotide exchange factor for eukaryotic initiation factor 2 (eIF2). eIF2B is a heteropentameric protein composed of alpha- subunits. The alpha, beta, and delta subunits form a regulatory subcomplex, while the gamma and form a catalytic subcomplex. Archaea possess homologues of alpha, beta, and delta subunits of eIF2B. Here, we report the three-dimensional structure of an archaeal regulatory subunit (aIF2Balpha) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 determined by X-ray crystallography at 2.2A resolution. aIF2Balpha consists of two subdomains, an N-domain (residues 1-95) and a C-domain (residues 96-276), connected by a long alpha-helix (alpha5: 78-106). The N-domain contains a five helix bundle structure, while the C-domain folds into the alpha/beta structure, thus showing similarity to D-ribose-5-phosphate isomerase structure. The presence of two molecules in the crystallographic asymmetric unit and the gel filtration analysis suggest a dimeric structure of aIF2Balpha in solution, interacting with each other by C-domains. Furthermore, the crystallographic 3-fold symmetry generates a homohexameric structure of aIF2Balpha; the interaction is primarily mediated by the long alpha-helix at the N-domains. This structure suggests an architecture of the three subunits, alpha, beta, and delta, in the regulatory subcomplex within eIF2B.

Amino Acid Sequence↗
PubMed2004

Crystal structure of the ribonuclease P protein Ph1877p from hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of pre-tRNA. Protein Ph1877p is one of essential components of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 RNase P [Biochem. Biophys. Res. Commun. 306 (2003) 666]. The crystal structure of Ph1877p was determined at 1.8A by X-ray crystallography and refined to a crystallographic R factor of 22.96% (Rfree of 26.77%). Ph1877p forms a TIM barrel structure, consisting of ten alpha-helices and seven beta-strands, and has the closest similarity to the TIM barrel domain of Escherichia coli cytosine deaminase with a root-mean square deviation of 3.0A. The protein Ph1877p forms an oblate ellipsoid, approximate dimensions being 45Ax43Ax39A, and the electrostatic representation indicated the presence of several clusters of positively charged amino acids present on the molecular surface. We made use of site-directed mutagenesis to assess the role of twelve charged amino acids, Lys42, Arg68, Arg87, Arg90, Asp98, Arg107, His114, Lys123, Lys158, Arg176, Asp180, and Lys196 related to the RNase P activity. Individual mutations of Arg90, Arg107, Lys123, Arg176, and Lys196 by Ala resulted in reconstituted particles with reduced enzymatic activities (32-48%) as compared with that reconstituted RNase P by wild-type Ph1877p. The results presented here provide an initial step for definite understanding of how archaeal and eukaryotic RNase Ps mediate substrate recognition and process 5'-leader sequence of pre-tRNA.

Amino Acid Sequence↗
PubMed2004

Structure of macrophomate synthase.

Macrophomate synthase (MPS) is an enzyme that catalyzes an extraordinarily complex conversion reaction, including two decarboxylations, two carbon-carbon bond formations and a dehydration, to form the benzoate analogue macrophomate from a 2-pyrone derivative and oxalacetate. Of these reactions, the two carbon-carbon bond formations are especially noteworthy because previous experiments have indicated that they proceed via a Diels-Alder reaction, one of the most widely used reactions in organic synthesis. The structural evidence that MPS catalyzes an intermolecular Diels-Alder reaction has been reported recently [Ose et al. (2003), Nature (London), 422, 185-189]. Interestingly, the tertiary structure as well as the quaternary structure of MPS are similar to those of 2-dehydro-3-deoxygalactarate (DDG) aldolase, a carbon-carbon bond-forming enzyme that catalyzes the reversible reaction of aldol condensation/cleavage. Here, the structure of MPS is described in detail and compared with that of DDG aldolase. Both enzymes have a (beta/alpha)(8)-barrel fold and are classified as belonging to the enolase superfamily based on their reaction strategy. The basic principles for carbon-carbon bond formation used by both MPS and DDG aldolase are the same with regard to trapping the enolate substrate and inducing subsequent reaction. The major differences in the active sites between these two enzymes are the recognition mechanisms of the second substrates, 2-pyrone and DDG, respectively.

Aldehyde-Lyases↗
PubMed2004

How oligomerization contributes to the thermostability of an archaeon protein. Protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii.

To study how oligomerization may contribute to the thermostability of archaeon proteins, we focused on a hexameric protein, protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii (StoPIMT). The crystal structure shows that StoPIMT has a distinctive hexameric structure composed of monomers consisting of two domains: an S-adenosylmethionine-dependent methyltransferase fold domain and a C-terminal alpha-helical domain. The hexameric structure includes three interfacial contact regions: major, minor, and coiled-coil. Several C-terminal deletion mutants were constructed and characterized. The hexameric structure and thermostability were retained when the C-terminal alpha-helical domain (Tyr(206)-Thr(231)) was deleted, suggesting that oligomerization via coiled-coil association using the C-terminal alpha-helical domains did not contribute critically to hexamerization or to the increased thermostability of the protein. Deletion of three additional residues located in the major contact region, Tyr(203)-Asp(204)-Asp(205), led to a significant decrease in hexamer stability and chemico/thermostability. Although replacement of Thr(146) and Asp(204), which form two hydrogen bonds in the interface in the major contact region, with Ala did not affect hexamer formation, these mutations led to a significant decrease in thermostability, suggesting that two residues in the major contact region make significant contributions to the increase in stability of the protein via hexamerization. These results suggest that cooperative hexamerization occurs via interactions of "hot spot" residues and that a couple of interfacial hot spot residues are responsible for enhancing thermostability via oligomerization.

Amino Acid Sequence↗
PubMed2004

Structure analysis of PH1161 protein, a transcriptional activator TenA homologue from the hyperthermophilic archaeon Pyrococcus horikoshii.

The crystal structure of the Bacillus subtilis TenA-homologue protein PH1161 from the hyperthermophilic archaebacterium Pyrococcus horikoshii was determined. TenA is known to belong to a new family of activators that stimulate the production of extracellular proteases in B. subtilis. A sequence-similarity search revealed that TenA-homologue proteins are widespread in bacteria and archaea, suggesting that this family of proteins plays an essential role in these organisms. In the present study, the first three-dimensional structure of a member of the TenA family of proteins was determined, unexpectedly revealing that the protein has a fold identical to that of haem oxygenase-1. Analysis has also shown that the protein has a unique ligand-binding pocket. Electron density of a bound ligand molecule was observed in this pocket. These results provide a valuable insight into the functional understanding of the TenA family of proteins.

Amino Acid Sequence↗