title: Identification and Characterization of a Novel Gentisate 1,2-Dioxygenase Gene from a Halophilic Martelella Strain.
abstract: Halophilic Martelella strain AD-3, isolated from highly saline petroleum-contaminated soil, can efficiently degrade polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene and anthracene, in 3-5% salinity. Gentisic acid is a key intermediate in the microbial degradation of PAH compounds. However, there is little information on PAH degradation by moderately halophilic bacteria. In this study, a 1,077-bp long gene encoding gentisate 1,2-dioxygenase (GDO) from a halophilic Martelella strain AD-3 was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme GDO was purified and characterized in detail. By using the (18)O isotope experiment and LC-MS analysis, the sources of the two oxygen atoms added onto maleylpyruvate were identified as H2O and O2, respectively. The Km and kcat values for gentisic acid were determined to be 26.64 μM and 161.29 s(-1), respectively. In addition, optimal GDO activity was observed at 30 °C, pH 7.0, and at 12% salinity. Site-directed mutagenesis demonstrated the importance of four highly conserved His residues at positions 155, 157, 167, and 169 for enzyme activity. This finding provides new insights into mechanism and variety of gentisate 1,2-dioxygenase for PAH degradation in high saline conditions.
authors.affiliation: State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China.
authors.forename: Ling
authors.initials: L
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
authors.forename: Haiyang
authors.initials: H
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Tang
authors.affiliation: State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China.
authors.forename: Yongdi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
authors.affiliation: State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Shi
authors.affiliation: State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China.
authors.forename: Kuangfei
authors.initials: K
authors.lastname: Lin
authors.affiliation: Shanghai Engineering Research Center of Contaminated Sites Remediation, Shanghai Institute for Design &Research in Environmental Engineering Co. Ltd., Shanghai 200232, People's Republic of China.
authors.forename: Qishi
authors.initials: Q
authors.lastname: Luo
authors.affiliation: State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China.
authors.forename: Changzheng
authors.initials: C
authors.lastname: Cui
mesh_terms: D020561:Alphaproteobacteria / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D000873:Anthracenes / Q000378:metabolism*; D001483:Base Sequence; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular; D049308:Dioxygenases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D059392:Petroleum Pollution; D010616:Phenanthrenes / Q000378:metabolism*; D010867:Pimelic Acids / Q000737:chemistry; D054712:Salinity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000737:chemistry; D012987:Soil / Q000737:chemistry; D012988:Soil Microbiology; D012989:Soil Pollutants
chemical_list: D000873:Anthracenes; D010616:Phenanthrenes; D010867:Pimelic Acids; D012987:Soil; D012989:Soil Pollutants; C113957:maleylpyruvic acid; C031181:phenanthrene; D012965:Sodium Chloride; D049308:Dioxygenases; C026247:gentisate 1,2-dioxygenase; C034020:anthracene