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 0.29mMand 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 Mg2+, Ni2+, Ca2+, or Zn2+. 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 0.2mMCDHB 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.