Molecular Mechanism Of Pyrrolidine Pathway Of Nicotine Degradation By Pseudomonas Putida S16

Nicotine is a toxic alkaloid from tobacco and also an environmental pollutant in areas near to cigarette production facilities.When tobacco is burned,nicotine is able to be converted into tobacco-specific nitrosamines?TSNAs?through incomplete combustion.One of the most toxic TSNAs,4-methylnitrosamino-l-?3-pyridyl?-l-butanone?NNK?,can be generated from pseudooxynicotine?PN?through nitrosation.PN is an intermediate product in the nicotine degradation pathway of Pseudomonas putida S16.Thus,the transformation of PN is essential to detoxication of nicotine in vivo.Over the last decade,our group has well studied the pyrrolidine pathway gene cluster nic2 of nicotine degradation in Pseudomonas putida S16.The function of most genes from nic2 has been identified through knocking-out genes and purification of the coded proteins in vitro.The nicotine degrading pathway has been unraveled by identifying the intermediate metabolites.Our lab has reported that the pnao gene is able to convert PN,which was confirmed by knocking-out genes.However,the molecular mechanism of the pnao gene is still unknown.Besides,the regulatory mechanism of P.putida S16 has not been uncovered comprehensively.The previous report was describing about the regulation of the distal genes in gene cluster nic2;however the regulatory mechanism of other genes in this cluster is still unknown.In this study,I focused on the function of pnao gene first.In previous study,our group knocked out pnao gene and identified the intermediate metabolites in the mutant strain.We believe pnao gene is responsible for transforming PN to3-succinoylpyridine?SP?,through removing the CH₃NH₂ group of PN.After this step,all of the intermediates in nicotine degrading pathway are unable to be converted to TSNAs,which makes the pnao gene significant for the detoxification of nicotine.Here,I characterize the properties and biochemical mechanism of the coded enzyme Pnao.Isotope labeling experiments provide a direct evidence that the newly introduced oxygen atom in 3-succinoylsemialdehyde-pyridine is derived from H₂O,but not from O₂.Pnao was very stable at temperatures below 50°C;below this temperature,the enzyme activity increased as the temperature rising.Site-directed mutagenesis studies showed that residue pro180 is important for its thermal stability.The pyrrolidine pathway of nicotine degradation in Pseudomonas putida S16 has been fully uncovered.However,little is known regarding whole mechanism?s?regulating transcription of the nicotine degradation pathway gene cluster.In the present study,I comprehensively elucidate an overall view of the NicR2-mediated two-step mechanism regulating 3-succinoyl-pyridine?SP?biotransformation,which involves the association of free NicR2 with two promoters and the dissociation of NicR2 from the NicR2-promoter complex.NicR2 can bind to another promoter Pspm,and regulate expression of the nicotine degrading genes in the middle of nic2 gene cluster,which are not controlled by the previously reported Phsp promoter.I identified the function of the inverted repeat based on the two promoters responsible for NicR2 binding,and found out that the-35/-10 motif for RNA polymerase is overlapped by the NicR2 binding site.I clarify the exact role of6-hydroxy-3-succinoyl-pyridine?HSP?,which acts as an antagonist and may prevent binding of free NicR2 to the promoters,but cannot release NicR2 from the promoters.Finally,a regulatory model is proposed,which consists of three parts:the interaction between NicR2 and two promoters?Pspm and Phsp?;the interaction between NicR2and two effectors?HSP and SP?;the interaction between NicR2 and RNA polymerase.Based on this model,I did some research on synthetic biology.I reconstructed a suicide system with plasmid pUCP18K-R-Phsp-ccdB,which was potential to be a wide-host suicide system.I also reconstructed a nicotine biosensor,which worked well in the nicotine concentration range between 0 and 5 g/L.To inhibit the background activity of wildtype Phsp promoter,I mutated this promoter by adding more DNA binding sites of NicR2,and the background activity of the mutant promoter was obviously inhibited.In this study,I identified the function of the pnao gene,uncovered the regulatory mechanism of the nicotine degrading cluster nic2,and tried to reconstruct the suicide system and nicotine biosensor based on this regulatory mechanism.This study extends our understanding of molecular mechanisms in biodegradation of environmental pollutants and toxicants.