Reaction mechanism based on X-ray crystallography at atomic resolution of endopolygalacturonase
| Project/Area Number |
15580072
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Hirosaki University |
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Principal Investigator |
MIYAIRI Kazuo Hirosaki University, Faculty of Agriculture and Life Sciences, Professor, 農学生命科学部, 教授 (10003526)
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| Co-Investigator(Kenkyū-buntansha) |
KATO Hiroaki Kyoto University, Graduate School of Pharmaceutical Science, Professor, 薬学研究科, 教授 (90204487)
YOSHIDA Takashi Hirosaki University, Faculty of Agriculture and Life Sciences, Associate Professor, 農学生命科学部, 助教授 (80200997)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | endopolygalacturonase / Stereum purpureum / general acid or base catalysis / X ray crystallography / atomic resolution / pectinase / 一般酸・塩基触媒 / 原子分解能X線結晶構造解析 |
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| Research Abstract |
○Expresion, purification and crystallization of Sterum purpureum endopolygalacturonase I in Escherichia coli.
We constructed an expression system, using Escherichia coli, and a purification method to prepare functionally active endoPG I for the mutation and crystallographic studies. Expression in E.coli strain Origami (DE3) provided soluble and active enzyme with proper disulfide bonds, whereas the enzyme expressed in BL21(DE3) was localized in inclusion bodies. Sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography.
○Purification, characterization and amino acid sequence of endopolygalacturonase IVa and IVb from Sterum purpureum.
To analysisi function of endoplygalacrturonase I we traied purification, characterization and amino acid sequence analysis of endopolygalacturonase IV, which have difference isoelectoric point and is produced by the same fungus Stereum purpureum. Two EndoPG IV, VIa and VIb were purified to homogeneity by three steps of column chromatography from the culture filtrate. The molecular masses of the EndoPG VIa and VIb were determined to be 36,017Da and 37,266Da using ESI-MS, respectively. These PGs have the common primary structure and one and two of sugar chain, respectively. By cloning of the cDNA with 5'-race and 3'race, the ammo acid sequence was decided. In this EndoPG IV, there are no deletion of C-terminal region (44 amino acid residues) observed in mature EndoPG I. The EndoPG IV was 72 % homology for amino acid sequences of mature EndoPG I.
○Determination of general basic catalysis by X ray crystallography at pH 2.5.
The pKa_1 and pKa_2 of the enzyme were determined at 4.35 and 5.3, respectively. Therefore, we tried to determine general basic catalysis by Xray crystallography at pH2.5 by for low. As the results, Asp153 seems to be the base catalyst candidate.
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Report
(3 results)
Research Products
(8 results)
