Mechanism of 4-Nitrophenol Oxidation in Rhodococcus sp Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression
Mechanism of 4-Nitrophenol Oxidation in Rhodococcus sp Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression
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DOI：
10.1128/jb.00742-08
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发表时间：
2008-11-01
影响因子：
3.2
通讯作者：
Negoro, Seiji
中科院分区：
文献类型：
--
作者：
Takeo, Masahiro;Murakami, Masumi;Negoro, Seiji
4-硝基苯酚（4-NP）是敌百虫等有机磷农药在土壤中水解的有毒产物。红球菌属菌株PN 1通过4-硝基邻苯二酚（4-NC）降解4-NP以用作唯一的碳、氮和能源。先前从PN 1克隆的5-kb EcoRI DNA片段含有参与4-NP氧化的基因簇（nphRA 1A 2）。根据序列分析，该基因簇预期编码AraC/XyIS家族调节蛋白（NphR）和双组分4-NP羟化酶（NphA 1和NphA 2）。在红球菌属菌株中的转录测定显示，nphA 1的转录仅由4-NP（测试的几种酚类化合物）在nphR存在下诱导，所述nphR是组成型表达的。破坏nphR废除转录活性，这表明nphR编码的正调控蛋白。4-NP羟化酶的两个蛋白质，NphA 1和NphA 2，在大肠杆菌中独立表达，并通过离子交换层析或亲和层析纯化。纯化的NphA 2减少黄素腺嘌呤二核苷酸（FAD）与伴随的氧化的NADH，而纯化的NphA 1氧化4-NP成4-NC几乎定量的FAD，NADH，和NphA 2的存在下。该功能分析，除了序列分析，揭示了该酶系统属于双组分黄素扩散单加氧酶家族。4-NP羟化酶对苯酚和4-氯苯酚的氧化活性与4-NP相当，对3-NP和4-NC的氧化活性较弱。
4-Nitrophenol (4-NP) is a toxic product of the hydrolysis of organophosphorus pesticides such as parathion in soil. Rhodococcus sp. strain PN1 degrades 4-NP via 4-nitrocatechol (4-NC) for use as the sole carbon, nitrogen, and energy source. A 5-kb EcoRI DNA fragment previously cloned from PN1 contained a gene cluster (nphRA1A2) involved in 4-NP oxidation. From sequence analysis, this gene cluster is expected to encode an AraC/XyIS family regulatory protein (NphR) and a two-component 4-NP hydroxylase (NphA1 and NphA2). A transcriptional assay in a Rhodococcus strain revealed that the transcription of nphA1 is induced by only 4-NP ( of several phenolic compounds tested) in the presence of nphR, which is constitutively expressed. Disruption of nphR abolished transcriptional activity, suggesting that nphR encodes a positive regulatory protein. The two proteins of the 4-NP hydroxylase, NphA1 and NphA2, were independently expressed in Escherichia coli and purified by ion-exchange chromatography or affinity chromatography. The purified NphA2 reduced flavin adenine dinucleotide ( FAD) with the concomitant oxidation of NADH, while the purified NphA1 oxidized 4-NP into 4-NC almost quantitatively in the presence of FAD, NADH, and NphA2. This functional analysis, in addition to the sequence analysis, revealed that this enzyme system belongs to the two-component flavin-diffusible monooxygenase family. The 4-NP hydroxylase showed comparable oxidation activities for phenol and 4-chlorophenol to that for 4-NP and weaker activities for 3-NP and 4-NC.