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Senda M.,JBIC Research Institute | Tanaka H.,Shiga University of Medical Science | Ishida T.,Shiga University of Medical Science | Horiike K.,Shiga University of Medical Science | Senda T.,Japan National Institute of Advanced Industrial Science and Technology
Acta Crystallographica Section F: Structural Biology and Crystallization Communications | Year: 2011

d-Serine dehydratase purified from chicken kidney was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 and 2-propanol as precipitants. The crystal belonged to space group P422, with unit-cell parameters a = 105.0, c = 81.89 Å, and diffracted to 2.09 Å resolution. An attempt to solve the structure using the MAD method is in progress. © 2011 International Union of Crystallography. All rights reserved.

Senda M.,JBIC Research Institute | Yamamoto A.,Shiga University of Medical Science | Tanaka H.,Shiga University of Medical Science | Ishida T.,Shiga University of Medical Science | And 2 more authors.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications | Year: 2012

d-Aspartate oxidase (DDO) from porcine kidney was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as a precipitant. The crystal belonged to space group P2 1, with unit-cell parameters a = 79.38, b = 144.0, c = 80.46 Å, Β = 101.1°, and diffracted to 1.80 Å resolution. Molecular-replacement trials using the structure of human d-amino-acid oxidase, which is 42% identical in sequence to DDO, as a search model provided a satisfactory solution. © 2012 International Union of Crystallography All rights reserved.

Tanaka H.,Shiga University of Medical Science | Senda M.,JBIC Research Institute | Venugopalan N.,Argonne National Laboratory | Yamamoto A.,Shiga University of Medical Science | And 3 more authors.
Journal of Biological Chemistry | Year: 2011

D-Serine is a physiological co-agonist of the N-methyl-D-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, D-amino acid oxidase degrades D-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a D-serine dehydratase degrades D-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated D-threonine aldolases, which are fold-type III pyridoxal 5′-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of D-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography.The enzyme activity on D-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn 2+.Noneof the reaction products that would be expected from side reactions of the PLP-D-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity.Wehave determined the first crystal structure of the D-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His 347 and Cys 349 is located near the PLP-Lys 45 Schiff base. A theoretical model of the enzyme-D-serine complex suggested that the hydroxyl group of D-serine directly coordinates the zinc ion, and that the ε-NH 2 group of Lys 45 is a short distance from the substrate Cα atom. The α-proton abstraction from D-serine by Lys 45 and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.

Ishikawa K.,Tohoku University | Ohsumi T.,Tohoku University | Tada S.,Tohoku University | Natsume R.,JBIC Research Institute | And 6 more authors.
Genes to Cells | Year: 2011

The nucleosome, which is composed of DNA wrapped around a histone octamer, is a fundamental unit of chromatin and is duplicated during the eukaryotic DNA replication process. The evolutionarily conserved histone chaperone cell cycle gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is involved in histone transfer and nucleosome reassembly during DNA replication. CIA/Asf1 has been reported to split the histone (H3-H4) 2 tetramer into histone H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication, CIA/Asf1 is involved in nucleosome disassembly and the promotion of semi-conservative histone H3-H4 dimer deposition onto each daughter strand in vivo. Despite numerous studies on the functional roles of CIA/Asf1, its mechanistic role(s) remains elusive because of lack of biochemical analyses. The biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks histone (H3-H4) 2 tetramer splitting activity, does not form efficiently a quaternary complex with histones H3-H4 and the minichromosome maintenance 2 (Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in histone transfer during DNA replication, negatively regulates the progression of the replication fork. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Maruyama Y.,Japan National Institute of Advanced Industrial Science and Technology | Ebihara T.,Japan National Institute of Advanced Industrial Science and Technology | Nishiyama H.,JEOL Ltd. | Konyuba Y.,JEOL Ltd. | And 5 more authors.
International Journal of Molecular Sciences | Year: 2012

X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called "crystallization bottleneck". Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few μm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

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