Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33
Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33
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DOI：
10.1128/aem.03909-15
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发表时间：
2016-03-01
影响因子：
4.4
通讯作者：
Wang, Shuning
中科院分区：
文献类型：
--
作者：
Li, Huili;Xie, Kebo;Wang, Shuning
尼古丁是烟草废物中的一种主要有毒生物碱，主要通过吡啶和吡咯烷途径被细菌降解。在此之前，我们在农杆菌S33中发现了一个新的吡啶和吡咯烷途径的杂交体，并对其关键酶6-羟基-3-琥珀酰吡啶（HSP）羟化酶进行了表征。在此，我们从菌株中纯化了初始化尼古丁降解的尼古丁脱氢酶，发现它与一种新的6-羟基假多氧尼古丁氧化酶形成复合物。纯化的复合物由ndhAB和pno编码的三个不同亚基组成，其中ndhA和ndhB重叠4 bp，距离pno约26 kb。根据基因序列和化学分析预测，NdhA （82.4 kDa）和NdhB （17.1 kDa）分别含有一个钼钼辅助因子和两个[2Fe-2S]簇，而Pno （73.3 kDa）含有一个黄素单核苷酸和一个[4Fe-4S]簇。ndhA或ndhB基因断裂的突变体在尼古丁上不能生长，但在6-羟基尼古丁和HSP上生长良好，而pno突变体在尼古丁和6-羟基尼古丁上不能生长，但在HSP上生长良好，这表明ndhA和ndhB参与了尼古丁氧化的初始化。我们成功地在大肠杆菌中表达了pno，发现重组pno在与6-羟基尼古丁氧化偶联时具有2,6-二氯酚吲哚酚还原活性。对纯化酶或突变体催化反应产物的测定表明，NdhAB催化尼古丁氧化生成6-羟基尼古丁，而Pno催化6-羟基假多羟基尼古丁氧化生成6-羟基-3-琥珀酰半醛吡啶。这些结果为烟叶杆菌S33的尼古丁降解新途径提供了新的见解。
Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are similar to 26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33.