Biomedical subjects

Takashi Kumasaka

Publications and source records attributed to Takashi Kumasaka.

At least 19 recordsLinked to original sources

PubMed2006

Cloning and functional characterization of the copper/zinc superoxide dismutase gene from the heavy-metal-tolerant yeast Cryptococcus liquefaciens strain N6.

The deep-sea yeast Cryptococcus liquefaciens strain N6 possesses high superoxide dismutase (SOD) activity and a high tolerance toward metal ions. To clarify the relationship between metal tolerance and SOD activity in this strain, we cloned the Cu/Zn SOD gene. This gene (Cl-SOD1) consists of 471 bp encoding 157 amino acids; the associated protein had 59.9-76.7% identity with Cu/Zn SOD proteins of other yeast species. The highest identity corresponded to Cryptococcus gattii (76.7%). Cl-SOD1 expression in the sod1 mutant of Saccharomyces cerevisiae revealed that this SOD protein was functional in S. cerevisiae. The Cl-SOD1 protein possessed approximately fourfold greater activity than S. cerevisiae SOD1 (Sc-SOD1) at 30 degrees C. The amount of Cl-SOD1 mRNA in strain N6 increased in the presence of copper ion. However, the level of this transcript was not dependent on an increase in copper ion concentration and did not correlate well with changes in the amount of Cu/Zn SOD protein. This result suggests that strain N6 possesses other Cu/Zn SOD genes induced in a manner different from Cl-SOD1 as found in Candida albicans, or that the Cl-SOD1 gene undergoes posttranscriptional regulation upon increase of copper ion.

Candida albicans↗

PubMed2006

Crystallization and preliminary X-ray diffraction studies of a hyperthermophilic Rieske protein variant (SDX-triple) with an engineered rubredoxin-like mononuclear iron site.

In place of the Rieske [2Fe-2S] cluster, an archetypal mononuclear iron site has rationally been designed into a hyperthermophilic archaeal Rieske [2Fe-2S] protein (sulredoxin) from Sulfolobus tokodaii by three residue replacements with reference to the Pyrococcus furiosus rubredoxin sequence. The resulting sulredoxin variant, SDX-triple (H44I/A45C/H64C), has been purified and crystallized by the hanging-drop vapour-diffusion method using 65%(v/v) 2-methyl-2,4-pentanediol, 0.025 M citric acid and 0.075 M sodium acetate trihydrate pH 4.3. The crystals diffract to 1.63 A resolution and belong to the triclinic space group P1, with unit-cell parameters a = 43.56, b = 76.54, c = 80.28 A, alpha = 88.12, beta = 78.82, gamma = 73.46 degrees. The asymmetric unit contains eight protein molecules.

Amino Acid Sequence↗

PubMed2006

The 1.48 A resolution crystal structure of the homotetrameric cytidine deaminase from mouse.

Cytidine deaminase (CDA) is a zinc-dependent enzyme that catalyzes the deamination of cytidine or deoxycytidine to form uridine or deoxyuridine. Here we present the crystal structure of mouse CDA (MmCDA), complexed with either tetrahydrouridine (THU), 3-deazauridine (DAU), or cytidine. In the MmCDA-DAU complex, it clearly demonstrates that cytidine is distinguished from uridine by its 4-NH(2) group that acts as a hydrogen bond donor. In the MmCDA-cytidine complex, cytidine, unexpectedly, binds as the substrate instead of the deaminated product in three of the four subunits, and in the remaining subunit it binds as the product uridine. Furthermore, the charge-neutralizing Arg68 of MmCDA has also exhibited two alternate conformations, I and II. In conformation I, the only conformation observed in the other structurally known homotetrameric CDAs, Arg68 hydrogen bonds Cys65 and Cys102 to modulate part of their negative charges. However, in conformation II the side chain of Arg68 rotates about 130 degrees around the Cgamma-Cdelta bond and abolishes these hydrogen bonds. The lack of hydrogen bonding may indirectly weaken the zinc-product interaction by increased electron donation from cysteine to the zinc ion, suggesting a novel product-expelling mechanism. On the basis of known structures, structural analysis further reveals two subclasses of homotetrameric CDAs that can be identified according to the position of the charge-neutralizing arginine residue. Implications for CDA-RNA interaction have also been considered.

3-Deazauridine↗

PubMed2006

RIKEN structural genomics beamlines at the SPring-8; high throughput protein crystallography with automated beamline operation.

RIKEN Structural Genomics Beamlines, BL26B1 & BL26B2 at the SPring-8, have been constructed for the structural genomics research. The main feature of the beamline is full automation of the successive data collections to maximize the beam-time efficiency. The beamline optics adopted a standard design commonly used for the SPring-8 bending magnet beamlines. Beamline instruments are operated by centralized control system through the computer network to achieve the automatic operation. The core part of the beamline development is a sample management system composed of sample changer robots for laboratory and beamline, and a networked sample database. BL26B1 has started user operation, and the automatic operation with the sample management system has been implemented at BL26B2.

Crystallography, X-Ray↗

PubMed2006

Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the [2Fe-2S] center.

The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.

Amino Acid Sequence↗

PubMed2006

Structural insight of human DEAD-box protein rck/p54 into its substrate recognition with conformational changes.

Human rck/p54, a product of the gene cloned at the breakpoint of t(11; 14) (q23;q32) chromosomal translocation on 11q23 in B-cell lymphoma, is a member of the DEAD-box RNA helicase family. Here, the crystal structure of Nc-rck/p54, the N-terminal core domain of rck/p54, revealed that the P-loop in motif I formed a closed conformation, which was induced by Asn131, a residue unique to the RCK subfamily. It appears that ATP does not bind to the P-loop. The results of dynamic light scattering revealed to ATP-induced conformational change of rck/p54. It was demonstrated that free rck/p54 is a distended molecule in solution, and that the approach between N-terminal core and C-terminal domains for ATP binding would be essential when unwinding RNA. The results from helicase assay using electron micrograph, ATP hydrolytic and luciferase assay showed that c-myc IRES RNA, whose secondary structure regulates IRES-dependant translation, was unwound by rck/p54 and indicated that it is a good substrate for rck/p54. Over-expression of rck/p54 in HeLa cells caused growth inhibition and cell cycle arrest at G2/M with down-regulation of c-myc expression. These findings altogether suggest that rck/p54 may affect the IRES-dependent translation of c-myc even in the cells.

Adenosine Triphosphate↗

PubMed2005

Crystallization and preliminary crystallographic analysis of endo-1,3-beta-glucanase from alkaliphilic Nocardiopsis sp. strain F96.

Endo-1,3-beta-glucanase, an enzyme that hydrolyzes the 1,3-beta-glycosyl linkages of beta-glucan, belongs to the family 16 glycosyl hydrolases, which are widely distributed among bacteria, fungi and higher plants. Crystals of a family 16 endo-1,3-beta-glucanase from the alkaliphilic Nocardiopsis sp. strain F96 were obtained by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a = 34.59, b = 71.84, c = 39.67 A, beta = 90.21 degrees, and contained one molecule per asymmetric unit. The Matthews coefficient (VM) and solvent content were 1.8 A3 Da(-1) and 31.8%, respectively. Diffraction data were collected to a resolution of 1.3 A and gave a data set with an overall Rmerge of 6.4% and a completeness of 99.3%.

Actinomycetales↗

PubMed2005

Structural basis of the substrate subsite and the highly thermal stability of xylanase 10B from Thermotoga maritima MSB8.

The crystal structure of xylanase 10B from Thermotoga maritima MSB8 (TmxB), a hyperthermostable xylanase, has been solved in its native form and in complex with xylobiose or xylotriose at 1.8 A resolution. In order to gain insight into the substrate subsite and the molecular features for thermal stability, we compared TmxB with family 10 xylanase structures from nine microorganisms. As expected, TmxB folds into a (beta/alpha)8-barrel structure, which is common among the glycoside hydrolase family 10. The enzyme active site and the environment surrounding the xylooligosaccharide of TmxB are highly similar to those of family 10 xylanases. However, only two xylose moieties were found in its binding pocket from the TmxB-xylotriose complex structure. This finding suggests that TmxB could be a potential biocatalyst for the large-scale production of xylobiose. The result of structural analyses also indicated that TmxB possesses some additional features that account for its thermostability. In particular, clusters of aromatic residues together with a lack of exposed hydrophobic residues are characteristic of the TmxB structure. TmxB has also a significant number of ion pairs on the protein surface that are not found in other thermophilic family 10 xylanases.

Bacterial Proteins↗

PubMed2005

Crystallization and X-ray analysis of 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics.

A recombinant 2-deoxy-scyllo-inosose synthase from Bacillus circulans has been crystallized at 277 K using PEG 4000 as precipitant. The diffraction pattern of the crystal extends to 2.30 A resolution at 100 K using synchrotron radiation at the Photon Factory. The crystals are monoclinic and belong to space group P2(1), with unit-cell parameters a = 80.5, b = 70.4, c = 83.0 A, beta = 117.8 degrees. The presence of two molecules per asymmetric unit gives a crystal volume per protein weight (VM) of 2.89 A3 Da(-1) and a solvent constant of 57.4% by volume.

Aminoglycosides↗

PubMed2005

Beamline Scheduling Software: administration software for automatic operation of the RIKEN structural genomics beamlines at SPring-8.

Beamline Scheduling Software (BSS) has been developed for the schedule management and equipment control of the RIKEN Structural Genomics Beamlines I and II (BL26B1 and BL26B2 at SPring-8). The beamline operation is automated with a sample-changer robot and a database system. The experimental schedule is registered to BSS as a list of diffraction measurements for numbers of protein crystals. BSS implements the beamline operation communicating with beamline instruments through the computer network. The RIKEN Structural Genomics Beamline I (BL26B1) opened for users using BSS in October 2002, and the automatic operation was implemented at BL26B2 in October 2003.

Automation↗

PubMed2005

Crystal structures of RsbQ, a stress-response regulator in Bacillus subtilis.

Growth-limiting stresses in bacteria induce the general stress response to protect the cells against future stresses. Energy stress caused by starvation conditions in Bacillus subtilis is transmitted to the sigma(B) transcription factor by stress-response regulators. RsbP, a positive regulator, is a phosphatase containing a PAS (Per-ARNT-Sim) domain and requires catalytic function of a putative alpha/beta hydrolase, RsbQ, to be activated. These two proteins have been found to interact with each other. We determined the crystal structures of RsbQ in native and inhibitor-bound forms to investigate why RsbP requires RsbQ. These structures confirm that RsbQ belongs to the alpha/beta hydrolase superfamily. Since the catalytic triad is buried inside the molecule due to the closed conformation, the active site is constructed as a hydrophobic cavity that is nearly isolated from the solvent. This suggests that RsbQ has specificity for a hydrophobic small compound rather than a macromolecule such as RsbP. Moreover, structural comparison with other alpha/beta hydrolases demonstrates that a unique loop region of RsbQ is a likely candidate for the interaction site with RsbP, and the interaction might be responsible for product release by operating the hydrophobic gate equipped between the cavity and the solvent. Our results support the possibility that RsbQ provides a cofactor molecule for the mature functionality of RsbP.

Bacillus subtilis↗

PubMed2005

A norovirus protease structure provides insights into active and substrate binding site integrity.

Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-A resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.

3C Viral Proteases↗

PubMed2004

Crystallization and preliminary X-ray studies on the reaction center-light-harvesting 1 core complex from Rhodopseudomonas viridis.

The reaction center-light-harvesting 1 (RC-LH1) core complex is the photosynthetic apparatus in the membrane of the purple photosynthetic bacterium Rhodopseudomonas viridis. The RC is surrounded by an LH1 complex that is constituted of oligomers of three types of apoproteins (alpha, beta and gamma chains) with associated bacteriochlorophyll bs and carotenoid. It has been crystallized by the sitting-drop vapour-diffusion method. A promising crystal diffracted to beyond 8.0 A resolution. It belonged to space group P1, with unit-cell parameters a = 141.4, b = 136.9, c = 185.3 A, alpha = 104.6, beta = 94.0, gamma = 110.7 degrees. A Patterson function calculated using data between 15.0 and 8.0 A resolution suggested that the LH1 complex is distributed with quasi-16-fold rotational symmetry around the RC.

Bacterial Proteins↗

PubMed2004

Crystallization and preliminary X-ray crystallographic analysis of chitinase F1 (ChiF1) from the alkaliphilic Nocardiopsis sp. strain F96.

Chitinase F1 (ChiF1) isolated from the alkaliphilic Nocardiopsis sp. strain F96 is a family 18 chitinase that hydrolyzes chitin, an insoluble beta-1,4-linked polymer of N-acetylglucosamine. Crystals of recombinant ChiF1 with molecular weight of 33 000 Da were grown to a suitable size for X-ray structure analysis using 18%(w/v) polyethylene glycol 8000, 200 mM zinc acetate dehydrate and 100 mM sodium cacodylate buffer pH 6.5. Diffraction data were collected at SPring-8 and show that the crystals belong to the trigonal space group P3(1)12 or P3(2)12, with unit-cell parameters a = 56.0, c = 179.5 A, and diffract X-rays beyond 1.2 A resolution. Crystallographic analysis was carried out using the multiwavelength anomalous diffraction (MAD) method using zinc as the anomalous scatter. The binding of Zn atoms was clarified from the Bijvoet and dispersive Patterson functions, which gave prominent zinc-zinc self-vectors on the Harker section.

Actinomycetales↗

PubMed2004

Crystallization and preliminary X-ray diffraction studies of the hyperthermophilic archaeal sulredoxin having the unique Rieske [2Fe-2S] cluster environment.

The hyperthermophilic archaeal sulredoxin from Sulfolobus tokodaii is a water-soluble high-potential Rieske [2Fe-2S] protein with unique pH-dependent redox properties compared with its mesophilic homologues in cytochrome bc1/b6f complexes. The oxidized recombinant sulredoxin has been crystallized by the hanging-drop vapour-diffusion method using 30%(v/v) polyethylene glycol 400, 0.1 M cadmium chloride and 0.1 M sodium acetate pH 4.6. The crystals diffracted to beyond 2.0 A resolution and belong to the cubic space group F4(1)32, with unit-cell parameter a = 163.00 +/- 0.05 A. The asymmetric unit contains one sulredoxin molecule. Three-wavelength MAD data were collected.

Archaeal Proteins↗

PubMed2004

Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-Oxo-prostaglandin 13-reductase catalytic mechanism and a possible Src homology 3 domain binding loop.

The bifunctional leukotriene B(4) 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB(4) 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B(4) (LTB(4)), and 15-oxo-lipoxin A(4) (15-oxo-LXA(4)). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB(4) 12-HD/PGR activity. Here we report the crystal structure of the LTB(4) 12-HD/PGR, the binary complex structure with NADP(+), and the ternary complex structure with NADP(+) and 15-oxo-PGE(2). In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE(2) contains two chains, but only the omega-chain of 15-oxo-PGE(2) was defined in the electron density map in the ternary complex structure. The omega-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2'-hydroxyl group of nicotine amide ribose of NADP(+) and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE(2) is consistent with a broad substrate specificity of LTB(4) 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB(4) 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the omega-chain.

15-Oxoprostaglandin 13-Reductase↗

PubMed2003

Crystallization and X-ray analysis of the N-terminal core domain of a tumour-associated human DEAD-box RNA helicase, rck/p54.

The RCK gene was cloned on the basis of the t(11;14)(q23;q32) chromosome translocation observed in human B-cell lymphoma cell line RC-K8. This gene was found to be overexpressed in various kinds of tumours. The gene product, rck/p54, consisting of 472 amino-acid residues with molecular weight 53.2 kDa, belongs to the family of DEAD-box RNA helicases. Its ATP-dependent RNA-unwinding activity toward c-myc RNA molecules in vitro has recently been demonstrated. In the present study, limited proteolysis experiments of rck/p54 were used to truncate the N-terminal domain (residues 1-288; 31.8 kDa) of rck/p54, leading to successful crystallization of Nc-rck/p54, i.e. the N-terminal core domain (residues 70-288; 24.5 kDa) of rck/p54. Crystals of Nc-rck/p54 were grown to a size suitable for X-ray structure analysis using polyethylene glycol 3350 as the precipitant. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 65.5, b = 73.1, c = 84.8 A, and diffracts X-rays to beyond 2.0 A resolution.

Amino Acid Sequence↗

PubMed2003

Structural insight into modest binding of a non-PXXP ligand to the signal transducing adaptor molecule-2 Src homology 3 domain.

Although some exceptional motifs have been identified, it is well known that the PXXP motif is the motif of ligand proteins generally recognized by the Src homology 3 (SH3) domain. SH3-ligand interactions are usually weak, with ordinary KD approximately 10 microM. The structural basis for a tight and specific association (KD = 0.24 microm) between Gads SH3 and a novel motif, PX(V/I)(D/N)RXXKP, was revealed in a previous structural analysis of the complex formed between them. In this paper, we report the crystal structure of the signal transducing adaptor molecule-2 (STAM2) SH3 domain in complex with a peptide with a novel motif derived from a ligand protein, UBPY. The derived KD value for this complex is 27 microM. The notable difference in affinity for these parallel complexes may be explained because the STAM2 SH3 structure does not provide a specificity pocket for binding, whereas the Gads SH3 structure does. Instead, the structure of STAM2 SH3 is analogous to that of Grb2 SH3 which, in addition to normal PXXP ligands, has also been shown to moderately recognize the novel motif discussed herein. Thus, the extremely tight interaction observed between Gads SH3 and the novel motif is caused not by an innate ability of the novel motif but rather by an evolutionary change in the Gads SH3 domain. Instead, SH3 domains of STAM2 and Grb2 retain the moderate characteristics of recognizing their ligand proteins like other SH3 domains for appropriate transient interactions between signaling molecules.

Adaptor Proteins, Signal Transducing↗