title: Metabolic Pathway Involved in 6-Chloro-2-Benzoxazolinone Degradation by Pigmentiphaga sp. Strain DL-8 and Identification of the Novel Metal-Dependent Hydrolase CbaA.
abstract: 6-Chloro-2-benzoxazolinone (CDHB) is a precursor of herbicide, insecticide, and fungicide synthesis and has a broad spectrum of biological activity. Pigmentiphaga sp. strain DL-8 can transform CDHB into 2-amino-5-chlorophenol (2A5CP), which it then utilizes as a carbon source for growth. The CDHB hydrolase (CbaA) was purified from strain DL-8, which can also hydrolyze 2-benzoxazolinone (BOA), 5-chloro-2-BOA, and benzamide. The specific activity of purified CbaA was 5,900 U · mg protein(-1) for CDHB, with Km and kcat values of 0.29 mM and 8,500 s(-1), respectively. The optimal pH for purified CbaA was 9.0, the highest activity was observed at 55°C, and the inactive metal-free enzyme could be reactivated by Mg(2+), Ni(2+), Ca(2+), or Zn(2+) Based on the results obtained for the CbaA peptide mass fingerprinting and draft genome sequence of strain DL-8, cbaA (encoding 339 amino acids) was cloned and expressed in Escherichia coli BL21(DE3). CbaA shared 18 to 21% identity with some metal-dependent hydrolases of the PF01499 family and contained the signature metal-binding motif Q127XXXQ131XD133XXXH137 The conserved amino acid residues His288 and Glu301 served as the proton donor and acceptor. E. coli BL21(DE3-pET-cbaA) resting cells could transform 0.2 mM CDHB into 2A5CP. The mutant strain DL-8ΔcbaA lost the ability to degrade CDHB but retained the ability to degrade 2A5CP, consistent with strain DL-8. These results indicated that cbaA was the key gene responsible for CDHB degradation by strain DL-8.
  
  
  
  2-Benzoxazolinone (BOA) derivatives are widely used as synthetic intermediates and are also an important group of allelochemicals acting in response to tissue damage or pathogen attack in gramineous plants. However, the degradation mechanism of BOA derivatives by microorganisms is not clear. In the present study, we reported the identification of CbaA and metabolic pathway responsible for the degradation of CDHB in Pigmentiphaga sp. DL-8. This will provide microorganism and gene resources for the bioremediation of the environmental pollution caused by BOA derivatives.
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.|State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People's Republic of China.
authors.forename: Weiliang
authors.initials: W
authors.lastname: Dong
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.|College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Huang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Yicheng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Xianfeng
authors.initials: X
authors.lastname: Ye
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Zhoukun
authors.initials: Z
authors.lastname: Li
authors.affiliation: College of Food and Bioengineering, Henan University of Science and Technology, Luoyang, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People's Republic of China.
authors.forename: Jiangfeng
authors.initials: J
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China czl@njau.edu.cn.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
mesh_terms: D042401:Alcaligenaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D001583:Benzoxazoles / Q000378:metabolism*; D001711:Biotransformation; D003001:Cloning, Molecular; D017124:Conserved Sequence; D020536:Enzyme Activators / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008670:Metals / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: C000622979:6-chloro-2-benzoxazolinone; D001583:Benzoxazoles; D020536:Enzyme Activators; D008670:Metals; D006867:Hydrolases