Cloning and characterization of a gene cluster involved in the catabolism of p-nitrophenol from Pseudomonas putida DLL-E4
Cloning and characterization of a gene cluster involved in the catabolism of p-nitrophenol from Pseudomonas putida DLL-E4
复制标题
恶臭假单胞菌DLL-E4对硝基苯酚分解代谢相关基因簇的克隆和表征
DOI：
10.1016/j.biortech.2010.04.052
复制
发表时间：
2010-10-01
影响因子：
11.4
通讯作者：
Cui, Zhongli
中科院分区：
文献类型：
--
作者：
Shen, Wenjing;Liu, Weidong;Cui, Zhongli
克隆了恶臭假单胞菌DLL-E4的一个9.2kb的DNA片段，该片段编码对硝基苯酚（PNP）分解代谢途径的酶，并进行了序列测定。共发现10个开放阅读框（ORF），其中5个ORF得到功能验证。通过Ni-NTA层析将pnpA和pnpC基因产物纯化至均一。PnpA是黄素腺嘌呤二核苷酸依赖性的单组分PNP 4-单加氧酶，其在NADH和FAD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，可将BT转化为马来酰乙酸。利用含有对苯二酚（HQ）双加氧酶（PnpC 1）和pnpC 2的质粒pET-pnpC 1C 2，在大肠杆菌中表达了PnpC 1C 2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是一个lysR型调节基因。pnpR是pnp基因簇中参与HQ降解的正调控因子。这些结果表明，pop基因簇编码的途径参与恶臭假单胞菌DLL-E4中HQ和BT的降解。(C)2010爱思唯尔有限公司保留所有权利。
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 colt 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 pop gene cluster is involved in degradation of HQ and BT in P. putida DLL-E4. (C) 2010 Elsevier Ltd. All rights reserved.