Biochemical Mechanism Of Nicotine Degradation In Agrobacterium Tumefaciens S33

Nicotine is a major toxic alkaloid in tobacco and tobacco wastes,and causes people addicted to tobacco.If improperly treated,nicotine could cause a serious harm to both the environment and human health due to its good water solubility.Microorganisms have become the main group in the degradation of environmental pollutants in nature because they are various in types,rich in diversity of metabolism,have strong adaptability to the environment,and cause no secondary pollution.Agrobacterium tumefaciens S33 has strong ability to degrade nicotine via a novel hybrid of pyridine pathway and pyrrolidine pathway.In this work,we made a further study on biochemical mechanism of nicotine degradation in strain S33,which will provide the basic theory for the treatment of tobacco wastes and biotransformation of nicotine into valuable compounds.By re-sequencing the complete genome,we found that the genome of strain S33 contained two chromosomes,one circular chromosome(2.49 Mbp)and one linear chromosome(2.98 Mbp).The annotation of the genome showed that the genes ndhB,ndhA,hno,pno,and hsh,identified previously be involved in nicotine degradation,formed a large gene cluster with genes encoding transposases,conjugal transfer proteins,plasmid partitioning,and replication initiation proteins.This gene cluster was located on a gene island(GI)of the circular chromosome,and also contained other genes(iso,nfo,hpo,and ami)involved in the downstream in the hybrid pathway of nicotine degradation in strain S33.On the basis of the high quality complete genome sequence of strain S33,we reanalyzed the transcriptomic data of S33,which were previously obtained by sequencing the two cells samples cultured in the nicotine medium(S33Nic)and the glucose-ammonium medium(S33Glu),respectively.The results confirmed the expression levels of the genes in the nicotine-degrading gene cluster were up-regulated when cultured in nicotine medium than in the glucose-ammonium medium.Including the 9 genes that have been experimentally verified to participate in nicotine degradation,another sevel genes were found to have an increase transcriptional activities in the same gene cluster(S33Nic VS S33Glu),such as paz and ald.We predicted that they were likely to play roles in nicotine degradation.Then 6-hydroxypseudooxynicotine oxidase(Pno)was characterized after heterologous expression and purification,which was previously thought to catalyze the forth step in nicotine degradation in strain S33.The UV-visible absorption spectra of the purified Pno showed that Pno contained typical absorption peaks of flavin and iron sulfur cluster.Thin layer chromatography and high performance liquid chromatography analysis showed that Pno contained 1.2 mol FMN of per mol protein.The determination of iron content demonstrated per mol protein contained 3.3 mol Fe.This was consisted with the bioformatics prediction that one Pno bound one FMN and one[4Fe4S].The enzyme activity results confirmed Pno could catalyze the oxidization of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoyl-semialdehydepyridine with the specific activity of 32.2 U/mg using 2,6-dichlorophenolindophenol as electron acceptor,and the apparent Km for 6-hydroxypseudooxynicotine was determined to be 0.37 mM.In addition,the location and purification of NdhAB catalyzing the initial step of nicotine degradation in strain S33 was studied.To obtain the periplasmic fraction of S33,we treated the cells with high hypertonic solution,lysozyme,and EDTA.Then the cytoplasm and cell membrance fractions were separated by brief sonication and ultracentrifugation.The enzyme activity assay showed that around 66%NdhAB activity was located in the periplasmic fraction,while around 90%Pno and Hno activities existed in the cytoplasmic fractions.Further purification of NdhAB from the periplasmic fraction was achieved.The results of enzyme activity assay and LC-MS analysis of the purified NdhAB demonstrated that NdhAB was transferred into the periplasmic space after synthesis in the cytoplasm,while Pno and Hno functioned in the cytoplasm,which was identical to the results of bioinformatics prediction that there was a typical Tat signal peptide at the N terminus of NdhA.Interestingly,an ORF in the upstream of ndhAB was predicted to encode electron transferring protein pseudoazurin(Paz)and to habor a signal peptide at its N terminus.It is known that Paz is usually located in the periplasmic space in the bacteria.In consideration of the up-regulation of Paz gene in the nicotine medium,we predicted that Paz might function as the physiological electron acceptor of NdhAB in strain S3 3.Thus,Paz was heterologouly expressed in E.coli.The purification of recombinant Paz from the periplasmic fraction of recombinant E.coli verified that Paz is really transported into the periplasmic space after expressed in the cytoplasm.And its signal peptide was cleaved as showed by determing its N-terminal amino acid residues.The purified recombinant Paz showed the typical absorption peaks of copper-containing protein pseudoazurin at 450 nm,595 nm,and 770 nm,where A595nm/A280nm was determined to be around 0.24,and the molar extinction coefficient of the oxidized Paz was around 3.58 mM-1 cm-1 at 595 nm.In addition,per mol Paz bound one mol copper atom.The enzyme activity assay and LC-MS analysis showed that Paz can function as the physiological electron acceptor of NdhAB,which catalyzed nicotine oxidation into 6-hydroxynicotine with the specific activity as 110.5 U/mg.For the reaction,the stoichiometry of oxidized nicotine and reduced Paz was determined to be 1:2.2,in consistent with that one Paz bound one copper atom.The apparent Km values for Paz and nicotine were determined to be 3.61 ?M and 1.64 ?M,respectively.The 18O2-labelled H2O assay indicated that the oxygen atom in the product 6-hydroxynicotine of the nicotine hydroxylation reaction was derived from the water molecule.Based on these results,it is concluded that Paz function as the physiological electron acceptor of NdhAB in the periplasmic space of strain S33 during nicotine degradation.In summary,we resequenced the complete genome sequence of strain S33,and reanalyzed the transcriptomic data of strain S33 based on the high quality genome sequence in this study.According to the genomic and transcriptomic analysis,the biochemical mechanism of the novel hybrid pathway in strain S33 was studied by purification and characterization of the key enzymes.This is the first time to characterize the properties and the function of 6-hydroxypseudooxynicotine oxidase(Pno)and to report that nicotine dehydrogenase(NdhAB)is located in the periplasmic space.Most importantly,Paz was found to function as the physiological electron acceptor of NdhAB to catalyze the hydroxylation of nicotine.These findings provide new insights to the biochemical mechanism of the novle hybrid pathway in strain S33.