The Molecular Catabolic Mechanism Of Nicotine By Pseudomonas Sp. HZN6

Nicotine is a natural alkaloid produced by tobacco plants that was once used as pesticide and is essential to the tobacco industry. Tobacco smoking is the leading cause of premature mortality, with more than6million tobacco-related deaths per year worldwild. Bioremediation of nicotine pollution is recognized as a cost-effective and reliable method. Therefore, it has been an important research item for us to isolate microbial resource of high efficiency nicotine-degrading, explore the role of the degradation mechanism, and give full play to their degradation ability to remove the environment pollution. It has great theoretical significance and application value.This research aimed at isolating the bacteria that can be independent of other carbon sources and degrade nicotine, study their degrading characteristics of nicotine in different environments, and provided the theory base for the bioremediation of environment pollution with nicotine at biochemical and molecular level; the same time, we cloned the key enzyme gene using the mutant library and further researched on the expression, enzyme properties, catalytic mechanisms and so on, which would be helpful to illustrate the novel degradation mechanism.A novel nicotine-degrading Pseudomonas sp. strain, HZN6, was isolated from a pesticide-wastewater treatment facility in Hangzhou. The strain could grow on nicotine as its sole source of carbon, nitrogen and energy. The HZN6strain could degrade approximately100%of500mg/1nicotine within12h at pH7.0and30℃.The strain’s main intermediate metabolites were determined to be pseudooxynicotine （PN）,3-succinoyl-pyridine （SP） and6-hydroxy-3-succinoyl-pyridine （HSP） using UV/Vis, HPLC, LC/MS and GC/MS. We could confirm that the major degradation pathway of nicotine in Pseudomonas sp. HZN6is pyrrolidine pathway:nicotine, PN, SP, HSP.A mutant library of the Pseudomonas sp.HZN6strain was generated by conjugation. The E. coli SM10λpir-containing plasmid pSC123was used for the donor strain. A result of229mutants were selected and classed. Mutant type I was that neither used nicotine as sole source of carbon nor degraded it at all. Mutant type Ⅱ was that could not use nicotine as sole source of carbon but could convert it into a intermediate. Mutant type III was that could not use nicotine as sole source of carbon but could degrade it completely.A mutant strain N6ml was determined to fail to degrade SP. A4583-bp DNA fragment （GenBank accession numbers HQ832741） from mutant N6ml was obtained through self-formed adaptor PCR （SEFA-PCR） and analysed. The mutant gene orfC displays89%deduced amino-acid sequence identity with the sirA-like gene （sirA2, a sulfurtransferase homologue gene） of P. stutzeri A1501. The orfC-disrupted strain lost the ability to degrade SP, and the complementation strains with the orfC from the P. sp. HZN6and the sirA2（PP₁233） from P. putida KT2440recovered the degradation ability. Though the orfC-disrupted strain also lost the xanthine dehydrogenase activity, the effects of tungsten on the degradation of SP and hypoxanthine revealed that the hydroxylation of SP to HSP was not a xanthine dehydrogenase type. These results demonstrated that the orfC gene was essential for the SP metabolism involved in the nicotine metabolic pathway in the Pseudomonas sp. HZN6strain. In addition, the physical organisation of the orfABCD gene cluster is highly conserved for all fully sequenced members of the Pseudomonas genus.A mutant strain N6mC8was determined to fail to degrade PN. A3874-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR. Two open reading frames （designated pao and sap） were analyzed, and the deduced amino acid sequences shared29%identity with6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans and49%identity with an aldehyde dehydrogenase from Bartonella henselae. Both pao and sap were cloned and functionally expressed in recombinant Escherichia coli BL21. The pao gene encoded a novel pseudooxynicotine amine oxidase with noncovalently bound FAD and exhibited substrate specificity removing the methylamine from pseudooxynicotine with the formation of3-succinoylsemialdehyde-pyridine and hydrogen dioxide. The content of FAD was determined to be0.51±0.01mol of FAD per mole subunit （average of10determinations）. The stoichiometry of the pseudooxynicotine amine oxidase reaction is as follows:PN was oxidatively deaminated using the noncovalent cofactor FAD in a reaction consuming O2and H2O and producing SAP, methylamine and H2O2（Fig.6D）.The optimal pH of PNAO was observed to be approximately8.0, and the optimal temperature was35℃. The apparent Km and Kcat values for PN were determined to be0.247±0.019mM and151±14s-1, respectively. The catalytic efficiency （kcat/Km） of611mM-1·s-1. The enzymatic activity was strongly inhibited by Ag+, Co2+, Cu2+, and Hg2+. In contrast, Ca2+, Mg2+, Mn2+, Ni2+, and Zn2+had no significant effects on the enzyme. PNAO activity was specific for PN. The sap encoded a NADP+-dependent3-succinoylsemialdehyde-pyridine dehydrogenase that catalyzed the dehydrogenation of3-succinoylsemialdehyde-pyridine to3-succinoyl-pyridine. The enzyme active was confirmed by a coupled PNAO-SAPD reaction. SAPD also utilized formaldehyde and acetaldehyde as substrates. Genetic analyses indicated that the pao gene played an essential role in nicotine or pseudooxynicotine mineralization in strain HZN6, whereas the sap gene did not.Then SEFA-PCR was performed to amplify the upstream sequences of the pao gene. A3640-bp DNA fragment was amplified. The nox gene was considered to be a probable candidate for the transformation of nicotine to pseudooxynicotine.After transformation of the nox gene into the non-nicotine-degrading strain KT2440, a recombinant was obtained with nicotine degrading activity. The nox-disrupted mutant N6Anox lost the ability to utilize nicotine, but not PN, as a sole carbon source. Based on these results, we concluded that the nox gene is responsible for the transformation of nicotine into PN in the Pseudomonas sp. strain HZN6. Furthermore, the KT2440strain containing the nox gene displayed approximately equal oxidative activity toward the two enantiomers of nicotine, i.e., NOX showed no isomer selectivity.RT-PCR results showed that mRNAs from the nox gene were produced in both DH5a and KT2440. These results indicated that additional factors are involved in the nicotine degradation process that are present in the Pseudomonas strains but not in the E. coli.The tertiary structure for the NOX protein was generated by the1-TASSER software. The conserved FAD-binding motif （GxGxxG） is proposed to interact with FAD, and His456is proposed to bind nicotine. Site-directed mutagenesis of His456Arg and the deletion of FAD-binding motif （GxGxxG） indicated that these two sites are essential in the nicotine-degrading process.