Dataset Information

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-8?cbaA 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.

SUBMITTER: Dong W

PROVIDER: S-EPMC4959186 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Metabolic Pathway Involved in 6-Chloro-2-Benzoxazolinone Degradation by Pigmentiphaga sp. Strain DL-8 and Identification of the Novel Metal-Dependent Hydrolase CbaA.

Dong Weiliang W Wang Fei F Huang Fei F Wang Yicheng Y Zhou Jie J Ye Xianfeng X Li Zhoukun Z Hou Ying Y Huang Yan Y Ma Jiangfeng J Jiang Min M Cui Zhongli Z

Applied and environmental microbiology 20160630 14

<h4>Unlabelled</h4>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 · m ...[more]

PMID: 27208123

Similar Datasets

Project description:Carbofuran, a broad-spectrum systemic insecticide, has been extensively used for approximately 50 years. Diverse carbofuran-degrading bacteria have been described, among which sphingomonads have exhibited an extraordinary ability to catabolize carbofuran; other bacteria can only convert carbofuran to carbofuran phenol, while all carbofuran-degrading sphingomonads can degrade both carbofuran and carbofuran phenol. However, the genetic basis of carbofuran catabolism in sphingomonads has not been well elucidated. In this work, we sequenced the draft genome of Sphingomonas sp. strain CDS-1 that can transform both carbofuran and carbofuran phenol but fails to grow on them. On the basis of the hypothesis that the genes involved in carbofuran catabolism are highly conserved among carbofuran-degrading sphingomonads, two such genes, cehACDS-1 and cfdCCDS-1, were predicted from the 84 open reading frames (ORFs) that share ?95% nucleic acid similarities between strain CDS-1 and another sphingomonad Novosphingobium sp. strain KN65.2 that is able to mineralize the benzene ring of carbofuran. The results of the gene knockout, genetic complementation, heterologous expression, and enzymatic experiments reveal that cehACDS-1 and cfdCCDS-1 are responsible for the conversion of carbofuran and carbofuran phenol, respectively, in strain CDS-1. CehACDS-1 hydrolyzes carbofuran to carbofuran phenol. CfdCCDS-1, a reduced flavin mononucleotide (FMNH2)- or reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase, hydroxylates carbofuran phenol at the benzene ring in the presence of NADH, FMN/FAD, and the reductase CfdX. It is worth noting that we found that carbaryl hydrolase CehAAC100, which was previously demonstrated to have no activity toward carbofuran, can actually convert carbofuran to carbofuran phenol, albeit with very low activity.IMPORTANCE Due to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. This study revealed the genetic determinants of carbofuran degradation in Sphingomonas sp. strain CDS-1. We speculate that the close homologues cehA and cfdC are highly conserved among other carbofuran-degrading sphingomonads and play the same roles as those described here. These findings deepen our understanding of the microbial degradation mechanism of carbofuran and lay a foundation for the better use of microbes to remediate carbofuran contamination.

Project description:Acetamiprid, a chloronicotinyl neonicotinoid insecticide, is among the most commonly used insecticides worldwide, and its environmental fate has caused considerable concern. The compound 1-(6-chloropyridin-3-yl)-N-methylmethanamine (IM 1-4) has been reported to be the main intermediate during acetamiprid catabolism in microorganisms, honeybees, and spinach. However, the molecular mechanism underlying the hydrolysis of acetamiprid to IM 1-4 has not yet been elucidated. In this study, a novel amidase (AceAB) that initially hydrolyzes the C-N bond of acetamiprid to generate IM 1-4 was purified and characterized from the acetamiprid-degrading strain Pigmentiphaga sp. strain D-2. Based on peptide profiling of the purified AceAB and the draft genome sequence of strain D-2, aceA (372 bp) and aceB (2,295 bp), encoding the α and β subunits of AceAB, respectively, were cloned and found to be necessary for acetamiprid hydrolysis in strain D-2. The characteristics of AceAB were also systematically investigated. Though AceA and AceB showed 35% to 56% identity to the α and β subunits of the N,N-dimethylformamidase from Paracoccus aminophilus, AceAB was specific for the hydrolysis of acetamiprid and showed no activities to N,N-dimethylformamide or its structural analogs.IMPORTANCE Acetamiprid, among the top neonicotinoid insecticides used worldwide, is one of the most important commercial insecticides. Due to its extensive use, the environmental fate of acetamiprid, especially its microbial degradation, has caused considerable concern. Although the catabolic pathways of acetamiprid in microorganisms have been extensively studied, the molecular mechanisms underlying acetamiprid biodegradation (except for a nitrile hydratase) remain largely unknown, and the enzyme responsible for the biotransformation of acetamiprid into its main intermediate, IM 1-4, have not yet been elucidated. The amidase AceAB and its encoding genes, aceA and aceB, characterized in this study, were found to be necessary and specific for the initial hydrolysis of the C-N bond of acetamiprid to generate IM 1-4 in Pigmentiphaga sp. strain D-2. The finding of the novel amidase AceAB will greatly enhance our understanding of the microbial catabolism of the widely used insecticide acetamiprid at the molecular level.

Dataset Information

Metabolic Pathway Involved in 6-Chloro-2-Benzoxazolinone Degradation by Pigmentiphaga sp. Strain DL-8 and Identification of the Novel Metal-Dependent Hydrolase CbaA.

Publications

Metabolic Pathway Involved in 6-Chloro-2-Benzoxazolinone Degradation by Pigmentiphaga sp. Strain DL-8 and Identification of the Novel Metal-Dependent Hydrolase CbaA.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets