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J. Biol. Chem., Vol. 278, Issue 24, 21483-21492, June 13, 2003

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Characterization of a Novel Thermostable Mn(II)-dependent 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from a Polychlorinated Biphenyl- and Naphthalene-degrading Bacillus sp. JF8^*

Takashi Hatta  , Gouri Mukerjee-Dhar ¶, Jiri Damborsky ||, Hohzoh Kiyohara  and Kazuhide Kimbara ¶

From the Research Institute of Technology, Okayama University of Science, 401-1 Seki, Okayama 703-8232, Japan, ^¶ Environmental Biotechnology Laboratory, Railway Technical Research Institute, Kokubunji, Tokyo 185-8540, Japan, ^|| National Centre for Biomolecular Research Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic

A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enzyme was purified to homogeneity. The enzyme has a molecular mass of 125 ± 10 kDa and was composed of four identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85 °C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80 °C and 81 min at 75 °C, making it the most thermostable extradiol dioxygenase studied. Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0–4.8 manganese atoms per enzyme molecule. The EPR spectrum of BphC_JF8 exhibited g = 2.02 and g = 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II). The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol. The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H₂O₂, 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mM 2,3-dihydroxybiphenyl. A decrease in K_m accompanied an increase in temperature, and the K_m value of 0.095 µM for 2,3-dihydroxybiphenyl (at 60 °C) is among the lowest reported. The kinetic properties and thermal stability of the native and recombinant enzyme were identical. The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases. The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8. A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme.

Received for publication, October 7, 2002 , and in revised form, March 14, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB092521.

^* This work was supported in part by Grant-in-aid for Scientific Research 12660091 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Takashi Hatta Research Institute of Technology, Okayama University of Science, 401-1 Seki, Okayama 703-8232, Japan. Tel.: 81-86-278-9349; Fax: 81-278-5312; E-mail: thatta{at}po.harenet.ne.jp.

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