Pendimethalin Nitroreductase Is Responsible for the Initial Pendimethalin Degradation Step in Bacillus subtilis Y3
Pendimethalin Nitroreductase Is Responsible for the Initial Pendimethalin Degradation Step in Bacillus subtilis Y3
复制标题
二甲戊灵硝基还原酶负责枯草芽孢杆菌 Y3 中二甲戊灵的初始降解步骤
DOI：
10.1128/aem.01771-16
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发表时间：
2016-12-01
影响因子：
4.4
通讯作者：
Hong, Qing
中科院分区：
文献类型：
--
作者：
Ni, Hai-yan;Wang, Fei;Hong, Qing
二甲戊乐灵[N-（1-乙基丙基）-2，6-二硝基-3，4-二甲基苯胺]是一种选择性的二硝基苯胺类除草剂。据报道，几种真菌和细菌降解二甲戊灵，但参与这一过程的酶或基因尚未得到表征。硝化还原是二甲戊乐灵降解和解毒的起始步骤。在这项研究中，二甲戊灵硝基还原酶（PNR），负责二甲戊灵的硝基还原，是从二甲戊灵降解菌株枯草芽孢杆菌Y3纯化。通过与Y3菌株基因组草图中所有蛋白质的质量指纹图谱比较，鉴定出一个硝基还原酶蛋白，并将其编码基因命名为pnr。PNR是一种功能性同源二聚体，亚基分子量约为23 kDa。PNR还原二甲戊灵芳环的C-6硝基，得到2-硝基-6-氨基-N-（1-乙基丙基）-3，4-二甲基苯胺。PNR还可以催化其他三种主要的二硝基苯胺类除草剂的硝基还原，包括丁乐灵，安磺灵和氟乐灵。然而，还原的硝基的数目是两个而不是一个，这不同于通过PNR的二甲戊乐灵的硝基还原，这可能是由于两个硝基的化学结构的对称性。解毒试验表明，PNR还原二甲戊灵对酿酒酵母BY 4741的生长没有抑制作用，而二甲戊灵对酿酒酵母BY 4741的生长有明显的抑制作用，表明二甲戊灵具有PNR解毒作用。因此，PNR在二甲戊乐灵解毒应用中具有潜力。本文报道了二甲戊乐灵和二硝基苯胺类除草剂生物降解过程中涉及的一种酶（及相应的基因）。二甲戊乐灵[N-（1-乙基丙基）-2，6-二硝基-3，4-二甲基苯胺]是一种广泛使用的选择性苗前二硝基苯胺类除草剂，在环境中经常检出其残留。美国环境保护署（EPA）已将二甲戊灵列为持久性生物累积毒素。到目前为止，没有酶或基因参与二甲戊乐灵生物降解的报道。在本研究中，基因pnr，它编码的硝基还原酶PNR，负责硝基还原二甲戊灵，是克隆的二甲戊灵降解菌株枯草芽孢杆菌Y3。PNR还可以催化其他三种主要的二硝基苯胺类除草剂的硝基还原，包括丁乐灵，安磺灵和氟乐灵。PNR对二甲戊乐灵的还原作用可消除其对酿酒酵母BY 4741的毒性，表明PNR在二甲戊乐灵解毒中具有应用潜力。
ABSTRACT Pendimethalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine] is a selective preemergence dinitroaniline herbicide. Several fungi and bacteria have been reported to degrade pendimethalin, but the enzymes or genes involved in this process have not been characterized. Nitroreduction is the initial degradation and detoxification step for pendimethalin. In this study, a pendimethalin nitroreductase (PNR), responsible for the nitroreduction of pendimethalin, was purified from the pendimethalin-degrading strain Bacillus subtilis Y3. Based on a comparison of its mass fingerprints with all of the deduced proteins from the draft genome of strain Y3, a protein annotated as a nitroreductase was identified, and its corresponding encoding gene was termed pnr. PNR was a functional homodimer with a subunit molecular mass of approximately 23 kDa. PNR reduced the C-6 nitro group of the aromatic ring of pendimethalin, yielding 2-nitro-6-amino-N-(1-ethylpropyl)-3,4-xylidine. PNR could also catalyze the nitroreduction of three other major varieties of dinitroaniline herbicides, including butralin, oryzalin, and trifluralin. However, the number of reduced nitro groups was two instead of one, which differed from the nitroreduction of pendimethalin by PNR and which may be due to the symmetry in the chemical structures of the two nitro groups. A detoxification assay revealed that 2-nitro-6-amino-N-(1-ethylpropyl)-3,4-xylidine (PNR-reduced pendimethalin) showed no inhibitory effect on the growth of Saccharomyces cerevisiae BY4741, whereas pendimethalin showed an obvious inhibitory effect on its growth, indicating the detoxification effect of pendimethalin by PNR. Therefore, PNR has potential in pendimethalin detoxification applications. This report describes an enzyme (and corresponding gene) involved in the biodegradation of pendimethalin and dinitroaniline herbicides. IMPORTANCE Pendimethalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine] is a widely used selective preemergence dinitroaniline herbicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency (EPA) has classified pendimethalin as a persistent bioaccumulative toxin. To date, no enzymes or genes involved in pendimethalin biodegradation have been reported. In the present study, the gene pnr, which encodes the nitroreductase PNR, responsible for the nitroreduction of pendimethalin, was cloned from the pendimethalin-degrading strain Bacillus subtilis Y3. PNR could also catalyze the nitroreduction of three other major varieties of dinitroaniline herbicides, including butralin, oryzalin, and trifluralin. The reduction of pendimethalin by PNR might eliminate its toxicity against Saccharomyces cerevisiae BY4741, indicating the application potential of PNR in the detoxification of pendimethalin.