Characterization of a Novel Thermostable Mn(II)-dependent 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from a Polychlorinated Biphenyl- and Naphthalene-degrading Bacillus sp. JF8
| Autor: | Gouri Mukerjee-Dhar, Takashi Hatta, Kazuhide Kimbara, Hohzoh Kiyohara, Jiri Damborsky |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular Time Factors Catechols Bacillus Ligands Biochemistry Substrate Specificity chemistry.chemical_compound Dioxygenase Cloning Molecular Phylogeny Chelating Agents chemistry.chemical_classification Biphenyl Chromatography 0303 health sciences biology Temperature Hydrogen-Ion Concentration Recombinant Proteins Ethanolamines Spectrophotometry Oxygenases Electrophoresis Polyacrylamide Gel Protein Binding Stereochemistry Molecular Sequence Data Naphthalenes Dioxygenases 03 medical and health sciences Amino Acid Sequence Molecular Biology Edetic Acid 030304 developmental biology Manganese Catechol Binding Sites Sequence Homology Amino Acid Molecular mass 030306 microbiology Thermophile Biphenyl Compounds Electron Spin Resonance Spectroscopy Active site DNA Cell Biology Protein Structure Tertiary Kinetics Enzyme Models Chemical chemistry biology.protein Protein quaternary structure |
| Zdroj: | Journal of Biological Chemistry. 278:21483-21492 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m210240200 |
| Popis: | 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 degrees C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80 degrees C and 81 min at 75 degrees 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-dihydroxybiphenyl3-methylcatecholcatechol4-methylcatechol4-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 Km accompanied an increase in temperature, and the Km value of 0.095 microm for 2,3-dihydroxybiphenyl (at 60 degrees 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. |
| Databáze: | OpenAIRE |
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