1999 Fiscal Year Final Research Report Summary
The structure and function of a complex metalloflavoprotein
| Project/Area Number |
10044324
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Nippon Medical School |
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Principal Investigator |
NISHINO Takeshi NIPPON MEDICAL SCHOOL, BIOCHEMISTRY AND MOLECULAR BIOLOGY, Professor, 大学院・医学研究科, 教授 (40094312)
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| Co-Investigator(Kenkyū-buntansha) |
IWASAKI Toshio NIPPON MEDICAL SCHOOL, BIOCHEMISTRY AND MOLECULAR BIOLOGY, Lecturer, 医学部, 講師 (40277497)
OKAMOTO Ken NIPPON MEDICAL SCHOOL, BIOCHEMISTRY AND MOLECULAR BIOLOGY, Lecturer, 医学部, 講師 (60267143)
MATSUMURA Tomohiro NIPPON MEDICAL SCHOOL, BIOCHEMISTRY AND MOLECULAR BIOLOGY, Assistant, 医学部, 助手 (20297930)
NISHINO Tomoko NIPPON MEDICAL SCHOOL, BIOCHEMISTRY AND MOLECULAR BIOLOGY, Assistant, 医学部, 助手 (80075613)
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| Project Period (FY) |
1998 – 1999
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| Keywords | X-ray crystal structure / xanthine dehydrogenase / xnathine oxidase / flavoprotein / non-heme iron / molybdenum enzyme / active oxygen / superoxide anion |
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| Research Abstract |
The mammalian enzymes, which catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, are synthesized as the dehydrogenase form (XDH) but can be readily converted to the oxidase form (XO) by oxidation ofsulfhydryl residues or by proteolysis. XDH shows a preference for NAD+ reduction at the FAD reaction site, while XO exclusivery uses dioxygen as its substrate leading to the formation of superoxide anion and hydrogen peroxide. Provided proper precautions are used, this protein can also be isolated in its XDH form, which can then be converted to the form that exhibits oxidase behavior. Due to major improvements in the purification of the protein from fat globular membranes from cow's milk, we were able to grow diffraction-quality crystals of both the XDH and XO forms in complex with the inhibitor salicylate. We present the crystal structures of the dimericbovine milk xanthine oxidoreductase in its XDH form at 2.1Å and in its XO from at 2.5Å resolution. Comparison of the two molecular structures identifies the major changes that occur during the proteolyically induced XDH to XO transformation.
The overall dimensions of the dimer are 155Åx90Åx70Å. The monomer can be divided into three domains. The conversion of XDH to XO may occur either reversibly by modification of Cys 535 and Cys 992, or irreversibly by proteolytic cleavage after Lys 551. Given the generally high structural conservation between the two forms of the enzyme, we are still investigating potential explanations of how the information about the surface modifications is transmitted to the buried cofactor binding site resulting in the major shift in conformation of a loop and a reversal of the electrostatic potential of this site. We are confident, however, that the ongoing analysis of diffraction data of improved resolution will provide answers to this question.
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