Cloning and characterization of a gene cluster involved in the catabolism of p-nitrophenol from Pseudomonas putida DLL-E4.
A 9.2-kb DNA fragment encoding the enzymes of a p-nitrophenol (PNP) catabolic pathway from Pseudomonas putida DLL-E4 was cloned and sequenced. Ten open reading frames (ORFs) were found and five ORFs were functionally verified. The pnpA and pnpC gene products were purified to homogeneity by Ni-NTA chromatography. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase which converts p-nitrophenol to para-benzoquinone in the presence of NADH and FAD. PnpC is a 1,2,4-trihydroxybenzene (BT) 1,2-dioxygenase which converts BT to maleylacetate. The hydroquinone (HQ) dioxygenase (PnpC1C2) multi-component protein complex was expressed in Escherichia coli via plasmid pET-pnpC1C2 containing pnpC1 and pnpC2. This complex converts HQ to gamma-hydroxymuconic semialdehyde. pnpR is a lysR-type regulator gene. PnpR is a positive regulator involved in HQ degradation in pnp gene cluster. These results demonstrate that a pathway encoded by the pnp gene cluster is involved in degradation of HQ and BT in P. putida DLL-E4.
Shen
Wenjing
W
Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
Liu
Weidong
W
Zhang
Jing
J
Tao
Jian
J
Deng
Haihua
H
Cao
Hui
H
Cui
Zhongli
Z
D001673:Biodegradation, Environmental; D002396:Catechols / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D049308:Dioxygenases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014158:Transcription, Genetic
D002396:Catechols; D009596:Nitrophenols; D049308:Dioxygenases; C041894:hydroxyquinol 1,2-dioxygenase; C001833:4-nitrocatechol; C024836:4-nitrophenol