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PubMed | Kagawa University, Maruwa foods and Biosciences Inc., RIKEN and Ikeda Tohka Industries Co.
Type: Journal Article | Journal: Acta crystallographica. Section F, Structural biology communications | Year: 2015

An FAD-dependent glucose dehydrogenase (GDH) from Aspergillus terreus was purified and crystallized at 293K using the sitting-drop vapour-diffusion method. A data set was collected to a resolution of 1.6 from a single crystal at 100K using a rotating-anode X-ray source. The crystal belonged to space group P21, with unit-cell parameters a = 56.56, b = 135.74, c = 74.13, = 90.37. The asymmetric unit contained two molecules of GDH. The Matthews coefficient was calculated to be 2.2(3)Da(-1) and the solvent content was estimated to be 44%.


PubMed | Fukui University of Technology, Chiyoda Corporation, Japan Aerospace Exploration Agency, University of Fukui and 2 more.
Type: Journal Article | Journal: PloS one | Year: 2016

The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed -helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st /-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd /-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, -1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation.


PubMed | Showa University, Chiyoda Corporation, Japan Aerospace Exploration Agency, Maruwa foods and Biosciences Inc. and 2 more.
Type: | Journal: Scientific reports | Year: 2015

The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double -barrel fold and a recently identified regulatory -helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms.


PubMed | Japan Atomic Energy Agency, Ibaraki University, University of Miyazaki, Maruwa foods and Biosciences Inc. and 2 more.
Type: Journal Article | Journal: Journal of the American Chemical Society | Year: 2015

Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IX (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 N 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.


Tanaka H.,Confocal Science Inc. | Tsurumura T.,Osaka Bioscience Institute | Aritake K.,Osaka Bioscience Institute | Furubayashi N.,Maruwa foods and Biosciences Inc. | And 9 more authors.
Journal of Synchrotron Radiation | Year: 2011

Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein.


Yamaguchi K.,Osaka University | Okamoto N.,Osaka University | Tokuoka K.,Osaka University | Sugiyama S.,Osaka University | And 5 more authors.
Journal of Synchrotron Radiation | Year: 2011

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.


PubMed | University of Ryukyus, Ibaraki University, University of Hyogo, University of Tokyo and 3 more.
Type: Journal Article | Journal: Science advances | Year: 2015

Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the Newtons cradle-like proton relay pathway of the catalytic cycle. Amide-imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.


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 | Year: 2013

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.


Takahashi S.,Chiyoda Corporation | Ohta K.,Japan Aerospace Exploration Agency | Furubayashi N.,Maruwa foods and Biosciences Inc. | Yan B.,Chiyoda Corporation | And 8 more authors.
Journal of Synchrotron Radiation | Year: 2013

The Japan Aerospace Exploration Agency (JAXA) started a high-quality protein crystal growth project, now called JAXA PCG, on the International Space Station (ISS) in 2002. Using the counter-diffusion technique, 14 sessions of experiments have been performed as of 2012 with 580 proteins crystallized in total. Over the course of these experiments, a user-friendly interface framework for high accessibility has been constructed and crystallization techniques improved; devices to maximize the use of the microgravity environment have been designed, resulting in some high-resolution crystal growth. If crystallization conditions were carefully fixed in ground-based experiments, high-quality protein crystals grew in microgravity in many experiments on the ISS, especially when a highly homogeneous protein sample and a viscous crystallization solution were employed. In this article, the current status of JAXA PCG is discussed, and a rational approach to high-quality protein crystal growth in microgravity based on numerical analyses is explained.


Inaka K.,Maruwa foods and Biosciences Inc. | Tanaka H.,Chiyoda Corporation | Takahashi S.,Chiyoda Corporation | Sano S.,Japan Aerospace Exploration Agency | And 3 more authors.
Defect and Diffusion Forum | Year: 2012

It is believed that a microgravity environment may maintain ideal depletion zones of protein (PDZ) and impurity (IDZ) around growing crystals and may contribute to growing high-quality crystals. This can lead to an X-ray diffraction data collection of higher resolution with lower mosaicity, because of the better internal order and fewer defects in the crystals when compared to ground-grown crystals. The extent of these depletion zones are dependent on a competition between the diffusion of the molecules in the solution (indexed by the diffusion coefficient, D) and the adsorption of those into the growing crystal (indexed by the kinetic constant, β). If we use the D/β value as an index of the extent of PDZ and IDZ, a lower D/β value is ideal for maintaining PDZ and IDZ. Using experimental results, we could easily obtain the D/β value. When we combined the D/β value with the quality of protein crystals obtained in microgravity experiments provided by Japanese Space Agency (JAXA), we found that the effects of microgravity contributed to obtaining superior crystals especially if the D/β value was less than 3 mm. The numerical analysis of the PDZ and IDZ shows that the radius of the crystal (R) is also related to the PDZ and the IDZ. If the Rβ/D value is large, both the PDZ and the IDZ provide a filtration effect, but if the Rβ/D value is small, only the IDZ does. © (2012) Trans Tech Publications.

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