Rid Enhances the 6-Hydroxypseudooxynicotine Dehydrogenase Reaction in Nicotine Degradation by Agrobacterium tumefaciens S33
Rid Enhances the 6-Hydroxypseudooxynicotine Dehydrogenase Reaction in Nicotine Degradation by Agrobacterium tumefaciens S33
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DOI：
10.1128/aem.02769-20
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发表时间：
2021-01
影响因子：
4.4
通讯作者：
Jinmeng Shang;Xia Wang;Meng Zhang;Rongshui Wang;Chengjia Zhang;Haiyan Huang;Shuning Wang
中科院分区：
文献类型：
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作者：
Jinmeng Shang;Xia Wang;Meng Zhang;Rongshui Wang;Chengjia Zhang;Haiyan Huang;Shuning Wang
RID是一个参与代谢物损伤修复的蛋白质家族，广泛分布于不同的生物体中。在本研究中，我们发现属于Rid6亚家族的RID-NC促进了根癌农杆菌S33降解尼古丁的吡啶和吡咯烷混合途径中的6-羟基伪烟碱脱氢酶(PNO)反应。摘要根癌农杆菌S33通过吡啶和吡咯烷途径的混合途径降解尼古丁。6-羟基假烟碱脱氢酶(PNO)氧化6-羟基-3-琥珀酰半醛-吡啶为6-羟基-3-琥珀酰半醛-吡啶是该途径中N-杂环分解的重要步骤。尽管对PNO进行了表征，但对该反应的了解还不完全；已知的是，它以很高的速度开始，然后反应速度迅速下降，导致形成非常少量的产物。在这项研究中，我们推测在反应中产生了一种不稳定的亚胺中间体，该中间体对代谢是有毒的。我们发现，尼古丁降解基因簇中的一个基因编码的RID蛋白(命名为RID-NC)促进了反应。RID是一个广泛分布的具有多种功能的小蛋白家族，一些亚家族具有脱氨酶活性，以消除活性中间体亚胺的毒性。生化分析表明，RID-NC减轻了PNO反应中可能产生的亚胺中间体的毒性，在RID-NC的存在下，PNO保持了较高的活性，反应产物的量至少增加了5倍。RID-NC基因的中断导致菌株S33在尼古丁上生长缓慢。探讨了RID-NC对亚胺中间体的解毒机理。系统发育分析表明，RID-NC属于鲜为人知的Rid6亚家族。这些结果进一步加深了我们对尼古丁降解的生化机制的理解，并为Rid6亚家族蛋白的功能提供了新的见解。重要性RID是一个参与代谢产物损伤修复的蛋白质家族，广泛分布于不同的生物体中。在本研究中，我们发现属于Rid6亚家族的RID-NC促进了根癌农杆菌S33降解尼古丁的吡啶和吡咯烷混合途径中的6-羟基伪烟碱脱氢酶(PNO)反应。RID-NC对反应中产生的假定反应性亚胺中间体进行水解，以消除其对PNO的毒性。这一发现加深了我们对有毒的N-杂环芳香族化合物在微生物中代谢过程的理解。本研究证明RID家族蛋白除参与氨基酸代谢外，还参与N-杂环芳香族生物碱的代谢，Rid6亚家族蛋白与RIDA亚家族蛋白相似，也具有脱氨酶活性。报道了反应性亚胺对非吡哆醛-5‘-磷酸依赖酶的损伤能力。这项研究为RID蛋白家族的功能提供了新的见解。
Rid is a family of proteins that participate in metabolite damage repair and is widely distributed in different organisms. In this study, we found that Rid-NC, which belongs to the Rid6 subfamily, promoted the 6-hydroxypseudooxynicotine dehydrogenase (Pno) reaction in the hybrid of the pyridine and pyrrolidine pathways for nicotine degradation by Agrobacterium tumefaciens S33. ABSTRACT Agrobacterium tumefaciens S33 degrades nicotine through a hybrid of the pyridine and pyrrolidine pathways. The oxidation of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoyl-semialdehyde-pyridine by 6-hydroxypseudooxynicotine dehydrogenase (Pno) is an important step in the breakdown of the N-heterocycle in this pathway. Although Pno has been characterized, the reaction is not fully understood; what is known is that it starts at a high speed followed by a rapid drop in the reaction rate, leading to the formation of a very small amount of product. In this study, we speculated that an unstable imine intermediate that is toxic with regard to the metabolism is produced in the reaction. We found that a Rid protein (designated Rid-NC) encoded by a gene in the nicotine-degrading gene cluster enhanced the reaction. Rid is a widely distributed family of small proteins with various functions, and some subfamilies have deaminase activity to eliminate the toxicity of the reactive intermediate, imine. Biochemical analyses showed that Rid-NC relieved the toxicity of the presumed imine intermediate produced in the Pno reaction and that, in the presence of Rid-NC, Pno maintained a high level of activity and the amount of the reaction product was increase by at least 5-fold. Disruption of the rid-NC gene led to slower growth of strain S33 on nicotine. The mechanism of Rid-NC-mediated detoxification of the imine intermediate was discussed. A phylogenetic analysis indicated that Rid-NC belongs to the rarely studied Rid6 subfamily. These results further our understanding of the biochemical mechanism of nicotine degradation and provide new insights into the function of the Rid6 subfamily proteins. IMPORTANCE Rid is a family of proteins that participate in metabolite damage repair and is widely distributed in different organisms. In this study, we found that Rid-NC, which belongs to the Rid6 subfamily, promoted the 6-hydroxypseudooxynicotine dehydrogenase (Pno) reaction in the hybrid of the pyridine and pyrrolidine pathways for nicotine degradation by Agrobacterium tumefaciens S33. Rid-NC hydrolyzed the presumed reactive imine intermediate produced in the reaction to remove its toxicity on Pno. The finding furthers our understanding of the metabolic process of the toxic N-heterocyclic aromatic compounds in microorganisms. This study demonstrated that the Rid family of proteins also functions in the metabolism of N-heterocyclic aromatic alkaloids, in addition to the amino acid metabolism, and that Rid6-subfamily proteins also have deaminase activity, similar to the RidA subfamily. The ability of reactive imines to damage a non-pyridoxal-5′-phosphate-dependent enzyme was reported. This study provides new insights into the function of the Rid family of proteins.