J 2003

Cloning and characterization of a novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from a PCB and naphthalene degrading Bacillus sp. JF8

HATTA, Takashi, Gouri MUKERJEE-DHAR, Jiri DAMBORSKY, Hohzoh KIYOHARA, Kazuhide KIMBARA et. al.

Basic information

Original name

Cloning and characterization of a novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from a PCB and naphthalene degrading Bacillus sp. JF8

Authors

HATTA, Takashi (392 Japan), Gouri MUKERJEE-DHAR (392 Japan), Jiri DAMBORSKY (203 Czech Republic, guarantor), Hohzoh KIYOHARA (392 Japan) and Kazuhide KIMBARA (392 Japan)

Edition

Journal of Biological Chemistry, 2003, 0021-9258

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 6.482

RIV identification code

RIV/00216224:14310/03:00008775

Organization unit

Faculty of Science

UT WoS

000183354200023

Keywords in English

ENZYME; THERMOSTABILITY; PCB; BIODEGRADATION; CLONING

Tags

Tags

Reviewed
Změněno: 19/3/2010 10:54, prof. Mgr. Jiří Damborský, Dr.

Abstract

V originále

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 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 4 identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85 C and a pH optimum of 8.5. 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. ICP-MS analysis confirmed the presence of 4.0 to 4.8 manganese atoms per enzyme molecule. The ESR 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 Km accompanied an increase in temperature and the Km value of 0.095 uM 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 which preferentially cleave bicyclic substrates are missing in BphC_JF8. A 3-D homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure and stability of the enzyme.

Links

LN00A016, research and development project
Name: BIOMOLEKULÁRNÍ CENTRUM
Investor: Ministry of Education, Youth and Sports of the CR, Biomolecular Center