Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, People's Republic of China.
Bioresour Technol. 2010 Oct;101(19):7516-22. doi: 10.1016/j.biortech.2010.04.052. Epub 2010 May 13.
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.
从恶臭假单胞菌 DLL-E4 中克隆并测序了一段编码对硝基苯酚 (PNP) 分解途径的酶的 9.2kb DNA 片段。发现了十个开放阅读框 (ORF)，其中五个 ORF 得到了功能验证。PnpA 和 pnpC 基因产物通过 Ni-NTA 层析法纯化至均一性。PnpA 是一种黄素腺嘌呤二核苷酸依赖性单组分 PNP 4-单加氧酶，在 NADH 和 FAD 的存在下将对硝基苯酚转化为对苯醌。PnpC 是一种 1,2,4-三羟基苯 (BT) 1,2-双加氧酶，将 BT 转化为马来酸乙酰酯。通过含有 pnpC1 和 pnpC2 的质粒 pET-pnpC1C2 在大肠杆菌中表达 HQ 双加氧酶 (PnpC1C2) 多组分蛋白复合物。该复合物将 HQ 转化为γ-羟基粘康酸半醛。pnpR 是一种 lysR 型调节基因。PnpR 是参与 pnp 基因簇中 HQ 降解的正调控因子。这些结果表明，pnp 基因簇编码的途径参与了恶臭假单胞菌 DLL-E4 中 HQ 和 BT 的降解。
