State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, People's Republic of China.
Institute of Basic Medicine, Shandong First Medical University & Shandong Academy of Medical Science, Jinan, People's Republic of China.
Appl Environ Microbiol. 2021 Mar 11;87(7). doi: 10.1128/AEM.02769-20.
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 -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 -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. 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 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 -heterocyclic aromatic compounds in microorganisms. This study demonstrated that the Rid family of proteins also functions in the metabolism of -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.
S33 通过吡啶和吡咯烷途径的混合降解尼古丁。6-羟基假氧烟碱脱氢酶（Pno）将 6-羟基-伪氧烟碱氧化为 6-羟基-3-琥珀酰半醛-吡啶，是该途径中 -杂环分解的重要步骤。尽管已经对 Pno 进行了表征，但该反应尚未完全了解；已知的是，它开始时速度很快，然后反应速度迅速下降，导致产物的形成量非常少。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产物，这种中间产物在代谢方面具有毒性。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）增强了该反应。Rid 是一种广泛分布的小蛋白家族，具有多种功能，某些亚家族具有脱氨酶活性，可以消除反应性中间产物亚胺的毒性。生化分析表明，Rid-NC 缓解了 Pno 反应中产生的假定亚胺中间产物的毒性，并且在 Rid-NC 的存在下，Pno 保持了高水平的活性，并且反应产物的量至少增加了 5 倍。-NC 基因的破坏导致 S33 在尼古丁上的生长速度变慢。讨论了 Rid-NC 介导的亚胺中间产物解毒的机制。系统发育分析表明，Rid-NC 属于研究甚少的 Rid6 亚家族。这些结果进一步加深了我们对尼古丁降解生化机制的理解，并为 Rid6 亚家族蛋白的功能提供了新的见解。Rid 是一种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。在这项研究中，我们发现属于 Rid6 亚家族的 Rid-NC 促进了 S33 中吡啶和吡咯烷途径混合降解尼古丁的 6-羟基假氧烟碱脱氢酶（Pno）反应。Rid-NC 将反应中产生的假定反应性亚胺中间产物水解，以去除其对 Pno 的毒性。这一发现进一步加深了我们对微生物中有毒 -杂环芳香化合物代谢过程的理解。本研究表明，Rid 蛋白家族除了参与氨基酸代谢外，还参与 -杂环芳香生物碱的代谢，并且 Rid6 亚家族蛋白也具有脱氨酶活性，类似于 RidA 亚家族。有报道称，反应性亚胺能够破坏非吡哆醛-5'-磷酸依赖性酶。本研究为 Rid 蛋白家族的功能提供了新的见解。
