| Co-Investigator(Kenkyū-buntansha) |
ISHIDA Tetsuo Shiga University of Medical Science, Assistant Professor, 医学部, 助手 (10176191)
NAKAI Chieko Shiga University of Medical Science, Assistant Professor, 医学部, 助手 (30111892)
HORIIKE Kihachiro Shiga University of Medical Science, Associate Professor, 医学部, 助教授 (80089870)
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| Research Abstract |
Catechol dioxygenases are classified into two groups : intradiol and extradiol. We have previously determined the DNA sequence of an extradiol enzyme, catechol 2, 3-dioxygenase. In order to pursue comparative studies between various catechol dioxygenases, we have tried to determine the DNA sequence of intradiol enzymes.
1. we have studied the well known intradiol enzyme, catechol 1, 2dioxygenase and found for the first time the existence of 3 isozymes, au, ap, op, of this enzyme from Pseudomonas arvilla C-1. we also succeeded to clone the gene for this enzyme from Pseudomonas putida mt-2.
2. An enzyme which cleaves the benzene ring of 3, 5-dichlorocatechol has been purified to homogeneity for the first time from a Pseudoiwnad, grown with a herbicide, 2, 4-dichlorophenoxyacetic acid (2, 4-D), as the sole carbon source. The enzyme was found to be a nonheme ferric dioxygenase and to catalyze the intradiol cleavage of 3, 5-dichlorocatechol. Thus, the enzyme was designated as 3, 5-dichlorocatechol 1, 2dioxygenase. Judging from the substrate specificity, kinetic constants, and N-terminal amino acid sequence, the enzyme was distinct from other chlorocatechol dioxygenases reported previously. We have also succeeded to clone the gene for this enzyme and the determination of the DNA sequence is now underway.
3. For the elucidation of the active site structure of catechol 2, 3dioxygenase, we used o-nitrophenol, a competitive inhibitor, and found it a useful active site probe for this enzyme. Using this probe, a novel mode interaction between substrate or the analog and the enzyme has been proposed. Trials were made to substitute the iron cofactor in the active site of this enzyme by other metals, and to modify the enzyme by sulfhydryl (-SH) blocking reagents or by suicide substrata. Basic information has been obtained from these studies on this enzyme.
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