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Higashiura A.,Osaka University | Kurakane T.,Osaka University | Matsuda M.,Osaka University | Suzuki M.,Osaka University | And 7 more authors.
Acta Crystallographica Section D: Biological Crystallography

Recent technical improvements in macromolecular X-ray crystallography have significantly improved the resolution limit of protein structures. However, examples of high-resolution structure determination are still limited. In this study, the X-ray crystal structure of bovine H-protein, a component of the glycine cleavage system, was determined at 0.88 Å resolution. This is the first ultrahigh-resolution structure of an H-protein. The data were collected using synchrotron radiation. Because of limitations of the hardware, especially the dynamic range of the CCD detector, three data sets (high-, medium-and low-resolution data sets) were measured in order to obtain a complete set of data. To improve the quality of the merged data, the reference data set was optimized for merging and the merged data were assessed by comparing merging statistics and R factors against the final model and the number of visualized H atoms. In addition, the advantages of merging three data sets were evaluated. The omission of low-resolution reflections had an adverse effect on visualization of H atoms in hydrogen-omit maps. Visualization of hydrogen electron density is a good indicator for assessing the quality of high-resolution X-ray diffraction data. © 2010 International Union of Crystallography. Source

Yamaguchi K.,Osaka University | Okamoto N.,Osaka University | Tokuoka K.,Osaka University | Sugiyama S.,Osaka University | And 5 more authors.
Journal of Synchrotron Radiation

Old yellow enzyme (OYE) is an NADPH oxidoreductase which contains flavin mononucleotide as prosthetic group. The X-ray structures of OYE from Trypanosoma cruzi (TcOYE) which produces prostaglandin (PG) F2α from PGH2 have been determined in the presence or absence of menadione. The binding motif of menadione, known as one of the inhibitors for TcOYE, should accelerate the structure-based development of novel anti-chagasic drugs that inhibit PGF2α production specifically. Source

Higashiura A.,Osaka University | Ohta K.,Japan Aerospace Exploration Agency | Masaki M.,Japan Aerospace Exploration Agency | Sato M.,Japan Aerospace Exploration Agency | And 3 more authors.
Journal of Synchrotron Radiation

Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. Structural comparisons between high-and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method. Source

Unno M.,Ibaraki University | Ishikawa-Suto K.,Ibaraki University | Ishikawa-Suto K.,Japan Atomic Energy Agency | Kusaka K.,Ibaraki University | And 15 more authors.
Journal of the American Chemical Society

Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IXα (BV) by phycocyanobilin:ferredoxin oxidoreductase (PcyA) via two steps of two-proton-coupled two-electron reduction. We determined the neutron structure of PcyA from cyanobacteria complexed with BV, revealing the exact location of the hydrogen atoms involved in catalysis. Notably, approximately half of the BV bound to PcyA was BVH+, a state in which all four pyrrole nitrogen atoms were protonated. The protonation states of BV complemented the protonation of adjacent Asp105. The "axial" water molecule that interacts with the neutral pyrrole nitrogen of the A-ring was identified. His88 Nδ was protonated to form a hydrogen bond with the lactam O atom of the BV A-ring. His88 and His74 were linked by hydrogen bonds via H3O+. These results imply that Asp105, His88, and the axial water molecule contribute to proton transfer during PcyA catalysis. © 2015 American Chemical Society. Source

Okamoto N.,Osaka University | Yamaguchi K.,Osaka University | Mizohata E.,Osaka University | Tokuoka K.,Osaka University | And 6 more authors.
Journal of Biochemistry

Old yellow enzyme (OYE) is an NADPH oxidoreductase capable of reducing a variety of compounds. It contains flavin mononucleotide (FMN) as a prosthetic group. A ternary complex structure of OYE from Trypanosoma cruzi (TcOYE) with FMN and one of the substrates, p-hydroxybenzaldehyde, shows a striking movement around the active site upon binding of the substrate. From a structural comparison of other OYE complexed with 12-oxophytodienoate, we have constructed a complex structure with another substrate, prostaglandin H2 (PGH2), to provide a proposed stereoselective reaction mechanism for the reduction of PGH2 to prostaglandin F2α by TcOYE. © The Author 2011. Source

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