2003 Fiscal Year Final Research Report Summary
In vitro evolution of SOD by DNA shufihing
| Project/Area Number |
14571781
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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 basic dentistry
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| Research Institution | Matsumoto Dental University |
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Principal Investigator |
HIRAOKA B.yukihiro Matsumoto Dental University, Graduate School of Oral Medicine, Professor, 大学院・歯学独立研究科, 教授 (20097512)
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| Co-Investigator(Kenkyū-buntansha) |
OKUMURA Shigeki Matsumoto Dental University, Dept of Oral Biochemistry, Research assosiate, 歯学部, 助手 (80350825)
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| Project Period (FY) |
2002 – 2003
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| Keywords | Reactive oxygen / SOD / cambialistic / Metallo enzyme / Oxidoreductase / Porphyromanas gingivalis / Porphyromonas gingivalis / 歯周病原菌 |
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| Research Abstract |
Mn-superoxide dismutases (SODs), Fe-SODs and cambiahistic SODs, its include P gingivalis (P.g.) SOD, have a large degree of sequence homology and X-ray structural similarity: the metal atoms are commonly ligated by three histidine residues, an aspartic acid residues and solvent molecule. We have prepared the mutant of the P.g. SOD with conversions of amino acid residues located near the active site using site-directed mutagenesis and DNA shuffling techniques.
[RESULTS] We made the Mn-and Fe-type mutation, by site-directed mutagenesis and expressed as a soluble fusion protein coupled to maltose binding protein (MBP) using the expression vector pMAL-c2. Enzymes were reconstituted with Mn or Fe by using an acid-guanidine-HCl method and purified to homogeneous by a hyroxyapatite-HPLC. Gly155Thr mutant SOD, reconstituted with Fe and Mn, analyzed an x-ray structure of the metal-reconstituted enzymes, and measured some properties of the enzymes. We found that this mutation changes the metal-specific activity drastically from a cambiahistic type to close to Fe-specific type. This is the first successful report among Fe-and Mn-SOD family to change the metal-specific activity not only drastically but also by a site-directed mutagenesis other than active site or second sphere. DNA shuffling and screening with paraquart methods, which consisted of a combination of cycles of random mutations and selection steps on the enzyme, provided the appropriate host strain for screening and conditions for random mutations.
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Research Products
(4 results)
