Takeo Masahiro, Murakami Masumi, Niihara Sanae, Yamamoto Kenta, Nishimura Munehiro, Kato Dai-ichiro, Negoro Seiji
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan.
J Bacteriol. 2008 Nov;190(22):7367-74. doi: 10.1128/JB.00742-08. Epub 2008 Sep 19.
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/XylS 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.
4-硝基苯酚（4-NP）是土壤中对硫磷等有机磷农药水解产生的有毒产物。红球菌属菌株PN1可通过4-硝基邻苯二酚（4-NC）降解4-NP，并将其用作唯一的碳源、氮源和能源。先前从PN1中克隆的一个5 kb的EcoRI DNA片段包含一个参与4-NP氧化的基因簇（nphRA1A2）。通过序列分析，该基因簇预计编码一种AraC/XylS家族调节蛋白（NphR）和一种双组分4-NP羟化酶（NphA1和NphA2）。在红球菌菌株中进行的转录分析表明，在组成型表达的nphR存在下，nphA1的转录仅由4-NP（在测试的几种酚类化合物中）诱导。nphR的破坏消除了转录活性，表明nphR编码一种正调节蛋白。4-NP羟化酶的两种蛋白NphA1和NphA2在大肠杆菌中独立表达，并通过离子交换色谱或亲和色谱进行纯化。纯化的NphA2伴随着NADH的氧化还原了黄素腺嘌呤二核苷酸（FAD），而纯化的NphA1在FAD、NADH和NphA2存在的情况下几乎将4-NP定量氧化为4-NC。除序列分析外，这种功能分析表明该酶系统属于双组分黄素可扩散单加氧酶家族。4-NP羟化酶对苯酚和4-氯苯酚的氧化活性与对4-NP的氧化活性相当，对3-NP和4-NC的活性较弱。
