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Acta Cryst. (2014). A70, C349
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Elucidation of the three-dimensional structure of biomolecules is of great importance because the three-dimensional structure is closely related to biological functions. X-ray single-crystal analysis is powerful method to analyze the structure, but it is sometimes difficult to grow a crystal sufficiently large for conventional or even synchrotron single-crystal X-ray measurement. We recently reported on a magnetically oriented microcrystal array (MOMA) [1] that is a composite in which microcrystals are aligned three-dimensionally in polymer matrix. Microcrystals are suspended in an ultraviolet-curable monomer and rotated non-uniformly in a static magnetic field to achieve three dimensional crystal alignment. Then, the monomer is photopolymerized to maintain the achieved alignment. We have successfully demonstrated that X-ray single crystal structure determinations through MOMA are possible for low molecular weight compounds [2] as well as protein. [3] However, the method with MOMA has two drawbacks: (i) the sample microcrystals cannot be recovered from a MOMA, which is especially serious problem in case of proteins, and (ii) the alignment is deteriorated during the consolidation process, causing low resolution. In this study, we attempt to solve these problems. First, we use a water-soluble sol as microcrystalline media and consolidate the alignment by gelation, which makes the recovery of microcrystals possible. Second, a magnetically oriented microcrystal suspension (MOMS) is used for in-situ X-ray diffraction measurement, which makes the sample recovery possible and enhances the resolution. We use lysozyme as a model protein for both cases. The in-situ method with in-house X-ray diffractometer gave diffraction spots about 3.0 Å resolutions. We plan to perform the same experiment at SPring-8.

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Acta Cryst. (2014). A70, C469
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Glycosyltransferase from Geobacillus sp. (SAS) is expected to see wide use as a starch antistaling enzyme in food including bread and rice products. The enzyme is thought to transfer maltotriose (G3) unit into non-reducing ends of sarch with unknown likage except for usual alpha-1,4 linkage. SAS was crystallized by sitting drop vapar diffusion method in 14~28% PEG4000 (w/v), 10mM CaCl2, 0.1M NaAC at pH 4.6 and 20°C for 1 month. The obtained crystals belong to a space group of P6522 with cell dimensions of a = b = 112 and c = 320 Å. The crystals were soaked in various oligomaltosaccharides (G1, G2, G3, G4, G5 and G6) for 15 min before flash cooling. The diffraction data of each complex were collected at beam-lines of BL26B1, BL38B1 and BL44XU in SPring-8. The crystal data were collected with 97-99 % completeness and Rmerge of 0.07-0.09 up to 1.6-2.3 Å resolution. The structures were determined by molecular replacement with cyclodextrin glucanotransferase (CGTase, PDB 1CYG) as a search model and were refined with PHENIX. The refined models of SAS/sugars contain one molecule of SAS comprising 733 amino acid residues, 5-8 calcium ions, 543-1141 water molecules and several sugars with R = 0.15-0.19 and Rfree = 0.16-0.23 for the data up to 1.6-2.3 Å resolution. SAS has almost the same overall structure with the CGTase except for several loops in the catalytic domain A. They share a similar active site except for subsite +3 where the non-reducing ends of the oligosaccharides bind. G1 bound to subsite +3, indicating +3 site has the highest affinity to G1. Only G3 was found to bind at subsites +3 ~ +1 when G3, G5 and G6 were soaked, whereas G4 bound at subsites +3 ~ -1 when G4 was soaked. From the clear density map of the bound G4, the bound glucose residue at subsute -1 is found to have alpha-1,6 linkage, indicating the product of this transglucosidase.

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Acta Cryst. (2014). A70, C1662
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Mesorhizobium loti MAFF303099, a nitrogen-fixing symbiotic bacterium, harbors degradation pathway I for pyridoxine (PN); a free form of vitamin B6. Pyridoxine 4-oxidase (PNOX), a monomeric glucose-methanol-choline (GMC) oxidoreductase family enzyme, is the first enzyme in the pathway. It catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal. PNOX with a C-terminal His6 tag was overexpressed in E.coli JM109 cells and purified with a Ni-NTA agarose column and a QA52 column. The tertiary structures of PNOX and a complex of PNOX with pyridoxamine (PM), which is a substrate analog, were determined at 2.2 Å and at 2.1 Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. The FAD interacts with the PNOX protein through a network of hydrogen bonds, which are mainly found in the ribose and pyrophosphate moieties of the FAD molecule. The surface structure of PNOX molecule showed that it had an opening socket for access of substrates. The opening was followed by a tunnel that was linked to the active site cavity. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 Å from the N4' atom of PM. The activities of H460A and H462A mutant PNOXs were very low, and H460A/H462A double mutant PNOX exhibited no activity. His462 may act as a general base for abstraction of a proton from the 4'-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4' atom in PM is located at 3.2 Å, and the hydride ion from the C4' atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase family shows that Pro504 in PNOX corresponds to Asn or His of the conserved His-Asn or His-His pair in other GMC oxidoreductases.
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