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Biomedical subjects

Isao Tanaka

Publications and source records attributed to Isao Tanaka.

At least 19 recordsLinked to original sources
PubMed2005

The CGL2612 protein from Corynebacterium glutamicum is a drug resistance-related transcriptional repressor: structural and functional analysis of a newly identified transcription factor from genomic DNA analysis.

The emergence of antibiotic-resistant bacteria often causes serious clinical problems. The TetR family is one of the major transcription factor families that regulate expression of genes involved in bacterial antimicrobial resistance systems. CGL2612 protein is a transcription factor newly identified by genomic DNA analysis on Corynebacterium glutamicum, which belongs to the mycolic acid-containing Actinomycetales, including the well known pathogens Corynebacterium diphtheriae and Mycobacterium tuberculosis. Crystal structure analysis showed that the CGL2612 protein exhibits significant structural similarity to the multidrug resistance (MDR)-related transcription factor QacR from Staphylococcus aureus, despite poor amino acid sequence similarity between these proteins. Binding DNA sequence analysis of CGL2612 protein using the systematic evolution of ligands by the exponential enrichment (systematic evolution of ligands by exponential enrichment, or SELEX) method revealed that this protein is a new member of the TetR family, which regulates expression of the immediately upstream gene, cgl2611, probably encoding a major facilitator superfamily permease. Subsequent functional analyses confirmed a function of the CGL2612 as a transcriptional repressor responsible for the antimicrobial resistance system in C. glutamicum. The strategy used in the present study is one of the most convenient and powerful methods to analyze functionally unknown transcription factors, and the results obtained here will contribute to our understanding of the drug resistance mechanism not only in C. glutamicum but also in the related bacteria, C. diphtheriae and M. tuberculosis.

Amino Acid Sequence↗
PubMed2005

Crystal structure of a ribonuclease P protein Ph1601p from Pyrococcus horikoshii OT3: an archaeal homologue of human nuclear ribonuclease P protein Rpp21.

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the removal of 5' leader sequences from tRNA precursors (pre-tRNA). The human protein Rpp21 is essential for human RNase P activity in tRNA processing in vitro. The crystal structure of Ph1601p from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, the archaeal homologue of Rpp21, was determined using the multiple anomalous dispersion (MAD) method with the aid of anomalous scattering in zinc and selenium at 1.6 A resolution. Ph1601p comprises an N-terminal domain (residues 1-55), a central linker domain (residues 56-79), and a C-terminal domain (residues 80-120), forming an L-shaped structure. The N-terminal domain consists of two long alpha-helices, while the central and C-terminal domains fold in a zinc ribbon domain. The electrostatic potential representation indicates the presence of positively charged clusters along the L arms, suggesting a possible role in RNA binding. A single zinc ion binds the well-ordered binding site that consists of four Cys residues (Cys68, Cys71, Cys97, and Cys100) and appears to stabilize the relative positions of the N- and C-domains. Mutations of Cys68 and Cys71 or Cys97 and Cys100 to Ser destabilize the protein structure, which results in inactivation of the RNase P activity. In addition, site-directed mutagenesis suggests that Lys69 at the central loop and Arg86 and Arg105 at the zinc ribbon domain are strongly involved in the functional activity, while Arg22, Tyr44, Arg65, and Arg84 play a modest role in the activity.

Amino Acid Sequence↗
PubMed2005

Low p53 expression of acute myelocytic leukemia cells with t(8;21) chromosome abnormality: association with low p14(ARF) expression.

In this study, the mRNA expression of p14(ARF) in t(8;21)AML cells was found to be significantly lower than acute myelocytic leukemia (AML) cells without t(8;21) chromosome abnormality, which was concordant with previous observation by Linggi et al. that AML1-MTG8 represses the transcription of p14(ARF). Although p53 mRNA expression level of t(8;21)AML cells was not low, p53 protein expression was reduced in t(8;21)AML cells. Genotoxic damage by ionizing radiation did not induce p53 upregulation in t(8;21)AML cells. Since p14(ARF) has been demonstrated to inhibit p53 degradation by binding to MDM2, repression of p14(ARF) expression in t(8;21)AML may facilitate the degradation of p53 by MDM2. Low p14(ARF) in t(8;21)AML may also account for the absence of upregulation of p53 by ionizing radiation. Then, we have shown that p53 expression level was inversely correlated with S/G2/M population of cell cycle in AML cells. Most of the t(8;21)AML are considered to be in p53(low) S/G2/M(high). It is now widely known that formation of AML1-MTG8 by t(8;21) translocation is a very early event in leukemogenesis, and AML1-MTG8 alone might have limited proliferative potential. Then, secondary oncogenic events such as activated receptor tyrosine kinase (like c-kit mutation), is necessary to become full-blown leukemia. Low p53 protein expression and insufficient induction of p53 by genotoxic damage might increase the opportunity to obtain additional oncogenic events, since genome guard function of p53 does not work in t(8;21)AML cells.

Cell Cycle↗
PubMed2005

Molecular basis of alanine discrimination in editing site.

AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRNA(Ala). Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid l-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thr-30 hydroxyl group located near the beta-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gln-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans- and cis-editing of tRNA(Ala).

Acylation↗
PubMed2005

Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method.

A novel and easy crystal-mounting technique was developed for the sulfur SAD method using Cr Kalpha radiation (2.29 A). Using this technique, the cryo-buffer and cryoloop around the protein crystal can be removed before data collection in order to eliminate their X-ray absorption. The superiority and reproducibility of the data sets with this mounting technique were demonstrated using tetragonal hen egg-white lysozyme crystals. The structure of a novel protein, PH1109, from Pyrococcus horikoshii OT3 was solved using this technique. At the wavelength of Cr Kalpha radiation, the anomalous signal |DeltaF|/|F| of PH1109 is expected to be 1.72% as this protein of 144 residues includes four methionines and two cysteines. Sulfur SAD phasing was performed using SHELXD and SHELXE. In the case of the data set obtained using this novel crystal-mounting technique, 54.9% of all residues were built with side chains automatically by RESOLVE. On the other hand, only 16.0% were built with side chains for the data set collected using the standard cryoloop. These results indicated that this crystal-mounting technique was superior to the standard loop-mounting method for the measurement of small anomalous differences at longer wavelength and yielded better results in sulfur-substructure solution and initial phasing. The present study demonstrates that the sulfur SAD method with a chromium source becomes enhanced and more practical for macromolecular structure determination using the new crystal-mounting technique.

Absorption↗
PubMed2005

Crystal structure of archaeal toxin-antitoxin RelE-RelB complex with implications for toxin activity and antitoxin effects.

The Escherichia coli chromosome encodes toxin-antitoxin pairs. The toxin RelE cleaves mRNA positioned at the A-site in ribosomes, whereas the antitoxin RelB relieves the effect of RelE. The hyperthermophilic archaeon Pyrococcus horikoshii OT3 has the archaeal homologs aRelE and aRelB. Here we report the crystal structure of aRelE in complex with aRelB determined at a resolution of 2.3 A. aRelE folds into an alpha/beta structure, whereas aRelB lacks a distinct hydrophobic core and extensively wraps around the molecular surface of aRelE. Neither component shows structural homology to known ribonucleases or their inhibitors. Site-directed mutagenesis suggests that Arg85, in the C-terminal region, is strongly involved in the functional activity of aRelE, whereas Arg40, Leu48, Arg58 and Arg65 play a modest role in the toxin's activity.

Antitoxins↗
PubMed2005

Structure of the type I L-asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii at 2.16 angstroms resolution.

The crystal structure of the L-asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhA) was determined by the multiwavelength anomalous diffraction (MAD) method and was refined to a resolution of 2.16 angstroms with a crystallographic R factor and free R factor of 21.1 and 25.3%, respectively. This is the first report of the three-dimensional structure of a type I L-asparaginase. These enyzmes are known as cytosolic L-asparaginases with lower affinities for substrate than the type II L-asparaginases. Although the overall fold of PhA was closely related to the structure of the well characterized type II L-asparaginase, structural differences were also detected. PhA forms a homodimer that corresponds to half the homotetramer of type II L-asparaginases. Structure comparison at the active site reveals that most catalytic residues are conserved except for two residues that recognize the amino group of the substrate. Additionally, a remarkable structural difference is found in the so-called 'active-site flexible loop'. In PhA this loop is stabilized by beta-hairpin formation and by elaborate interactions with the type-I-specific alpha-helical region derived from the other subunit forming the PhA dimer. The flexible loop of the type II enzyme is considered to serve as a mobile gate to the active site. Therefore, the loop stabilization observed in the PhA structure may cause limitation of the access of the substrate to the active site.

Amino Acid Sequence↗
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↗