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Nobuo Okazaki

Publications and source records attributed to Nobuo Okazaki.

13 recordsLinked to original sources
PubMed2006

Structure of the stand-alone RAM-domain protein from Thermus thermophilus HB8.

The stand-alone RAM (regulation of amino-acid metabolism) domain protein SraA from Thermus thermophilus HB8 (TTHA0845) was crystallized in the presence of zinc ions. The X-ray crystal structure was determined using a multiple-wavelength anomalous dispersion technique and was refined at 2.4 A resolution to a final R factor of 25.0%. The monomeric structure is a betaalphabetabetaalphabeta fold and it dimerizes mainly through interactions between the antiparallel beta-sheets. Furthermore, five SraA dimers form a ring with external and internal diameters of 70 and 20 A, respectively. This decameric structure is unique compared with the octameric and dodecameric structures found for other stand-alone RAM-domain proteins and the C-terminal RAM domains of Lrp/AsnC-family proteins.

Amino Acid Sequence↗
PubMed2006

Evaluation of pulmonary function using breathing chest radiography with a dynamic flat panel detector: primary results in pulmonary diseases.

OBJECTIVES: Dynamic flat panel detectors (FPD) permit acquisition of distortion-free radiographs with a large field of view and high image quality. The present study was performed to evaluate pulmonary function using breathing chest radiography with a dynamic FPD. We report primary results of a clinical study and computer algorithm for quantifying and visualizing relative local pulmonary airflow. MATERIALS AND METHODS: Dynamic chest radiographs of 18 subjects (1 emphysema, 2 asthma, 4 interstitial pneumonia, 1 pulmonary nodule, and 10 normal controls) were obtained during respiration using an FPD system. We measured respiratory changes in distance from the lung apex to the diaphragm (DLD) and pixel values in each lung area. Subsequently, the interframe differences (D-frame) and difference values between maximum inspiratory and expiratory phases (D-max) were calculated. D-max in each lung represents relative vital capacity (VC) and regional D-frames represent pulmonary airflow in each local area. D-frames were superimposed on dynamic chest radiographs in the form of color display (fusion images). The results obtained using our methods were compared with findings on computed tomography (CT) images and pulmonary functional test (PFT), which were examined before inclusion in the study. RESULTS: In normal subjects, the D-frames were distributed symmetrically in both lungs throughout all respiratory phases. However, subjects with pulmonary diseases showed D-frame distribution patterns that differed from the normal pattern. In subjects with air trapping, there were some areas with D-frames near zero indicated as colorless areas on fusion images. These areas also corresponded to the areas showing air trapping on computed tomography images. In asthma, obstructive abnormality was indicated by areas continuously showing D-frame near zero in the upper lung. Patients with interstitial pneumonia commonly showed fusion images with an uneven color distribution accompanied by increased D-frames in the area identified as normal on computed tomography images. Furthermore, measurement of DLD was very effective for evaluating diaphragmatic kinetics. CONCLUSIONS: This is a rapid and simple method for evaluation of respiratory kinetics for pulmonary diseases, which can reveal abnormalities in diaphragmatic kinetics and regional lung ventilation. Furthermore, quantification and visualization of respiratory kinetics is useful as an aid in interpreting dynamic chest radiographs.

Adult↗
PubMed2005

Crystal structure of novel NADP-dependent 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8.

3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific dehydrogenase belonging to the 3-hydroxyacid dehydrogenase family. To gain insight into the function of this enzyme at the atomic level, we have determined the first crystal structures of the 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8: holo enzyme and sulfate ion complex. The crystal structures reveal a unique tetrameric oligomerization and a bound cofactor NADP+. This bacterial enzyme may adopt a novel cofactor-dependence on NADP, whereas NAD is preferred in eukaryotic enzymes. The protomer folds into two distinct domains with open/closed interdomain conformations. The cofactor NADP+ with syn nicotinamide and the sulfate ion are bound to distinct sites located at the interdomain cleft of the protomer through an induced-fit domain closure upon cofactor binding. From the structural comparison with the crystal structure of 6-phosphogluconate dehydrogenase, another member of the 3-hydroxyacid dehydrogenase family, it is suggested that the observed sulfate ion and the substrate 3-hydroxyisobutyrate share the same binding pocket. The observed oligomeric state might be important for the catalytic function through forming the active site involving two adjacent subunits, which seems to be conserved in the 3-hydroxyacid dehydrogenases. A kinetic study confirms that this enzyme has strict substrate specificity for 3-hydroxyisobutyrate and serine, but it cannot distinguish the chirality of the substrates. Lys165 is likely the catalytic residue of the enzyme.

Alcohol Oxidoreductases↗
PubMed2004

Purification, crystallization and preliminary crystallographic analysis of the glycine-cleavage system component T-protein from Pyrococcus horikoshii OT3.

The glycine-cleavage system component T-protein is a folate-dependent enzyme that catalyzes the formation of ammonia and 5,10-CH2-tetrahydrofolate from the aminomethyl intermediate bound to the lipoate cofactor of H-protein. T-protein from Pyrococcus horikoshii OT3 has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. X-ray diffraction data have been collected to 1.50 A resolution at 100 K using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 78.980, b = 95.708, c = 118.331 A. Assuming one homodimer per asymmetric unit gives a VM value of 2.4 A3 Da(-1) and a solvent content of 49.0%.

Aminomethyltransferase↗
PubMed2004

First determination of the inhibitor complex structure of human hematopoietic prostaglandin D synthase.

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD(2) as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 A resolution in the presence of Mg(2+). The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp104, inducing large movement around the alpha5-helix, which caused the space group of the complex crystal to change from P2(1) to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC(50) value of BSPT for human H-PGDS from 36.2 micro M to 98.1 micro M upon binding of Mg(2+), because the K(m) value of GSH for human H-PGDS was decreased from 0.60 micro M in the presence of EDTA to 0.14 micro M in the presence of Mg(2+). We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg(2+) in the mM range in the cytosol for further development of structure-based anti-allergic drugs.

Benzothiazoles↗
PubMed2003

Mechanism of metal activation of human hematopoietic prostaglandin D synthase.

Here we report the crystal structures of human hematopoietic prostaglandin (PG) D synthase bound to glutathione (GSH) and Ca2+ or Mg2+. Using GSH as a cofactor, prostaglandin D synthase catalyzes the isomerization of PGH2 to PGD2, a mediator for allergy response. The enzyme is a homodimer, and Ca2+ or Mg2+ increases its activity to approximately 150% of the basal level, with half maximum effective concentrations of 400 microM for Ca2+ and 50 microM for Mg2+. In the Mg2+-bound form, the ion is octahedrally coordinated by six water molecules at the dimer interface. The water molecules are surrounded by pairs of Asp93, Asp96 and Asp97 from each subunit. Ca(2+) is coordinated by five water molecules and an Asp96 from one subunit. The Asp96 residue in the Ca2+-bound form makes hydrogen bonds with two guanidium nitrogen atoms of Arg14 in the GSH-binding pocket. Mg2+ alters the coordinating water structure and reduces one hydrogen bond between Asp96 and Arg14, thereby changing the interaction between Arg14 and GSH. This effect explains a four-fold reduction in the K(m) of the enzyme for GSH. The structure provides insights into how Ca2+ or Mg2+ binding activates human hematopoietic PGD synthase.

Amino Acid Sequence↗