Investigation of the mechanism of D-ring inversion in porphyrin biosynthesis
| Project/Area Number |
14580658
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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 |
Functional biochemistry
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| Research Institution | Kurume University |
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Principal Investigator |
OMATA Yoshiaki Kurume University, School of Medicine, Associate Professor, 医学部, 助教授 (20268840)
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| Co-Investigator(Kenkyū-buntansha) |
NOGUCHI Masato Kurume University, School of Medicine, Professor, 医学部, 教授 (10124611)
SAKAMOTO Hiroshi Kurume University, School of Medicine, Lecturer, 医学部, 講師 (70309748)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2004: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
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| Keywords | prophyrin / uroporphyrinogen / uroporphyrinogen III syntase / congenital erythropoietic porphyria / hydroxymethylbilane / hydroxymethylbilane synthase |
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| Research Abstract |
The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen III synthase(UROS). Although the role of UROS is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of UROS have yet to be studied. An expression system in E.coli and a purification procedure for human UROS were established in this project. The enzyme in the lysate was unstable, but glycerol is able to prevent the activity loss in the lysate for at least a few days after lysis. The enzyme therefore was purified in the presence of glycerol, the entire procedure being completed within 48 hr after lysis with the aid of an HPLC system. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of accessible Cys residues and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human UROS, this Cys residue was considered to be Cys73, which is deeply buried inside the enzyme. A mutation of Cys73 to Arg is most common in congenital erythropoietic porphyria(CEP), in which UROS is deficient, having been found in the allele of 33% of CEP patients. These findings suggest that Cys73 of human UROS has an important role in catalysis. From the results of chemical modifications that indicate the role of several basic residues, Lys, Arg and His residues around putative active site were mutated. The activity and kinetic parameters of mutated enzymes suppose that substrate is bound between two opposite residues and surrounded by the other residues.
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Report
(4 results)
Research Products
(33 results)
