caa a22 4500 397494033 CHVBK 20180308164516.0 cr unu---uuuuu 161202e199503 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a124753 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a124753 Overexpression of Pseudomonas putida Catechol 2,3-Dioxygenase with High Specific Activity by Genetically Engineered Escherichia coli [Elektronische Daten] [Takehiro Kobayashi, Tetsuo Ishida, Kihachiro Horiike, Yoshiyuki Takahara, Naganori Numao, Atsushi Nakazawa, Teruko Nakazawa, Mitsuhiro Nozaki] The cloned xylE gene encoding catechol 2,3-dioxygenase (metapyrocatechase) from TOL plasmid in Pseudomonas putida mt-2 has been expressed in Escherichia coli W3110 to a level of ˜15% of the total soluble protein. Of the total iron in the crude extract, 45° was on the enzyme. The crystallized enzyme from E. coli had higher iron content (3.7 mol/mol enzyme) and specific activity (536 U/mg) than the enzyme from P. putida mt-2. However, no differences were observed in physicochemical, protein-chemical, and kinetic properties between the two enzymes. The enzyme was a homotetramer, and no changes were observed in the values of Mr (136,000 ±5,000) and Stokes radius (4.26 nm) in the concentration range from 0.36 nM to 2.8 μM, indicating that the native enzyme neither dissociated into subunits nor polymerized in this range. The catalytic center activity and the .Km values for catechol and dioxygen were 278 s−1 1.87 and 7.45 μM, respectively, at pH 7.5 and 25°C. The enzyme showed a broad substrate specificity. Among substrates, 4-methylcatechol and 4-chlorocatechol showed specificity constants (˜200 μM−1·s−1) higher than that for catechol. Acetone and phenol derivatives competitively inhibited the activity against catechol. The relationship between specific activity and iron content was not linear, suggesting some conformational changes in the partially iron-depleted enzyme. © 1995 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence catechol 2,3-dioxygenase nationallicence metapyrocatechase nationallicence nonheme iron dioxygenase nationallicence TOL plasmid nationallicence xylE expression nationallicence Kobayashi Takehiro Department of Biochemistry, Shiga University of Medical Science Seta, Ohtsu, Shiga 520-21 aut Ishida Tetsuo Department of Biochemistry, Shiga University of Medical Science Seta, Ohtsu, Shiga 520-21 aut Horiike Kihachiro Department of Biochemistry, Shiga University of Medical Science Seta, Ohtsu, Shiga 520-21 aut Takahara Yoshiyuki Tokyo Research Center, Central Glass Co., Ltd. Kawagoe, Saitama 350-11 aut Numao Naganori Sagami Chemical Research Center, 4-4-1 Nishi-Ohnuma Sagamihara, Kanagawa 229 aut Nakazawa Atsushi Departments of Biochemistry, Yamaguchi University School of Medicine Ube, Yamaguchi 755 aut Nakazawa Teruko Departments of Microbiology, Yamaguchi University School of Medicine Ube, Yamaguchi 755 aut Nozaki Mitsuhiro Department of Biochemistry, Shiga University of Medical Science Seta, Ohtsu, Shiga 520-21 aut The Journal of Biochemistry Oxford University Press 117/3(1995-03), 614-622 0021-924X 117:3<614 1995 117 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a124753 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a124753 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kobayashi Takehiro Department of Biochemistry, Shiga University of Medical Science Seta, Ohtsu, Shiga 520-21 aut NATIONALLICENCE 700 1- Ishida Tetsuo Department of Biochemistry, Shiga University of Medical Science Seta, Ohtsu, Shiga 520-21 aut NATIONALLICENCE 700 1- Horiike Kihachiro Department of Biochemistry, Shiga University of Medical Science Seta, Ohtsu, Shiga 520-21 aut NATIONALLICENCE 700 1- Takahara Yoshiyuki Tokyo Research Center, Central Glass Co., Ltd. Kawagoe, Saitama 350-11 aut NATIONALLICENCE 700 1- Numao Naganori Sagami Chemical Research Center, 4-4-1 Nishi-Ohnuma Sagamihara, Kanagawa 229 aut NATIONALLICENCE 700 1- Nakazawa Atsushi Departments of Biochemistry, Yamaguchi University School of Medicine Ube, Yamaguchi 755 aut NATIONALLICENCE 700 1- Nakazawa Teruko Departments of Microbiology, Yamaguchi University School of Medicine Ube, Yamaguchi 755 aut NATIONALLICENCE 700 1- Nozaki Mitsuhiro Department of Biochemistry, Shiga University of Medical Science Seta, Ohtsu, Shiga 520-21 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 117/3(1995-03), 614-622 0021-924X 117:3<614 1995 117 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford