Cloning And Functional Analysis Of Nornicotine Degradation Gene In Shinella Sp.HZN7

Nornicotine(NOR),a natural alkaloid,a precursor to the N'-nitrosonornicotine(NNN)that is believed to be one of tobacco-specific nitrosamine(TSNAs)carcinogen.It can directly induce abnormal glycosylation of protein in smokers' plasma,covalently react with commonly used steroids,affecting efficacy and toxicity.Therefore,the control of nornicotin content is far-reaching significance for the maintenance of human health.Therefore,it has been an important research item for us to isolate microbial resource of high efficiency nornicotine-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.The significance of this study is to use the existing nicotine-degrading bacteria as a research material to study the degradation of nornicotine.We determine the degradation pathway of nornicotine and cloned into a two-step key degrading enzyme gene,analyze the differences with nicotine by bioinformatics,reveal new mechanisms for the degradation of nornicotine from molecular biology,biochemistry and bioinformatics.This study provides a theoretical and technical basis for microbial degradation and green conversion of environmental pollution caused by nornicotine.In this paper,molecular biology,biochemistry and bioinformatics were used to study the degradation of nornicotine from the following aspects.A novel nicotine-degrading Shinella sp.HZN7,was isolated from a pesticide-wastewater treatment facility in Hangzhou.In the previous study,we determined that the pathways of this strain to degrade nicotine were variant of the pyridine and pyrrolidine pathways,constructed a library of nicotine degradation inactivated mutants by pSC123 transposon mutagenesis and photographic plates,and screened 323 mutants.In this study,we selected four representative mutant N7-W13,N7-M9,N7-X5 and N7-M17 from 323 mutants.They are responsible for the accumulation of intermediates Nicotine,6HN,6HPON and HSP,respectively,in the degradation process,that is,the first step,the second step,the third step and the fourth step of the degradation capacity.We used the strains HZN7,N7-W13,N7-M9,N7-X5 and N7-M17 to degrade nicotine and nornicotine,respectively.The results of UV spectra showed that the HZN7 could not completely mineralize nornicotine.and the UV peaks of N7-X5 and strain N7-M17 were consistent with the peak shape of strain HZN7,the degradation of nornicotine by strain N7-W13 produced a new peak shape that was not the same as the previous three strains,and the UV results of strain N7-W13 did not change.We initially speculated that in strain HZN7 the same group of genes was responsible for the degradation of nornicotine and degradation of nicotine.This group of genes is only responsible for the first two steps of normicotine degradation and presumably the first two steps of the product are 6-hydroxy-niacin(6HNor)and 6-hydroxy sarcosine(6HM).We analyzed the genotype of the strain HZN7 to clone the nornicotine degradation gene in strain HZN7.The genome of the strain Shinella sp.HZN7 was obtained using the PacBio RSII high-throughput sequencing technique.The results showed that the Shinella sp.HZN7 genome consisted of a complete chromosome sequence and twelve plasmid sequences(named pShin-01 to pShin-12).The GC content of whole genome was analyzed by R language.It was found that the GC content of pShin-05 was much lower than that of whole genome,and it was suspected that the plasmid was transferred from gene level.It is tentatively speculated that the nornicotine degradation gene is located on pShin-05.We used the Aligend Sequences function of Snapgene 2.3.2 software to compare the HZN7 genome sequence with the currently reported 6-hydroxy-nicotine oxidase NctB gene sequence.Aligned Sequences alignment results show that NctB is located on pShin-05.Since the biodegradable genes responsible for a particular compound are often located in a cluster of genes,the gene responsible for the first degradation step of demethylated nicotine is assumed to be adjacent to the nctB gene.Downstream of nctB gene,one ORF,named nctA2(2250 bp),encodes a 749 amino acid protein that harbors a conserved domain for molybdopterin cofactor.NCBI BLASTP searches showed that NctA2 exhibits 100%amino acid identity to the nicotine hydroxylase large subunit VppAL from strain Ochrobactrum sp.SJY1.Another ORF,named nctA1(468 bp),encodes a 155-amino acid protein that harbors a conserved domain for[2Fe-2S]clusters.NctAl exhibits 100%amino acid identity to the nicotine hydroxylase small-subunit VppAs from strain SJY1.Due to the homology between NctA1A2 and VppA,we considered NctA1 and NctA2 to be a two-component nicotine dehydrogenase and responsible for the initial hydroxylation step of nicotine to 6HN in Shinella sp.HZN7.We used molecular biology methods to verify the above gene function.The fragments containing NctA1A2 genes were cloned into the vector pBBR1MCS2 and were introduced into Pseudomonas putida KT2440 by triparental conjugative.Pseudomonas putida KT2440 which containing plasmid pBnctA1A2 have the ability to transform nornicotine.After transformation of the nctA1A2 gene into the non-nornicotine-degrading strain KT2440,a recombinant was obtained with nornicotine degrading activity.The nctA1A2-disrupted mutuan N7?nctA lost the ability to utilize nornicotine.Based on these results,we conclude that the nctA gene is responsible for the transformation of nornicotine into 6HNor in the Shinella sp.HZN7.6HNor was identified as 5-Pyrrolidin-2-yl-pyridin-2-ol by UV,LC/TOF-MS,GC/TOF-MS and NMR.The 6-hydroxy-nicotine oxidase NctB was cloned into pET 29a(+)and expressed in E.coli BL21(DE3).The recombinant proteins were purified by Ni-affinity chromatography.Enzymatic assays showed that enzyme NctB can convert 6HNor to 6HM.The nctB-disrupted mutuan N7?nctB lost the ability to utilize 6HNor.Based on these results,we conclude that the nctB gene is responsible for the transformation of 6HNor into 6HM in the Shinella sp.HZN7.6HM was identified as 5-(4,5-Dihydro-3H-pyrrol-2-yl)-pyridin-2-ol instead of 5-(4,5-Dihydro-1H-pyrrol-2-yl)-pyridin-2-ol by UV,LC/TOF-MS,GC/TOF-MS and NMR.That is,the dehydrogenation site of NctB-converted 6HNor is C7 and N11 on the pyrrole ring,and the dehydrogenation site of 6HN is C7 and C8 on the pyrrole ring.The structure and function information of 6-hydroxy-nicotine oxidase NctB secondary structure and active site were predicted by bioinformatics tools,which provided the necessary theoretical guidance for functional research.In the SWISS database,a 6HLO crystal model(5ttk.1),which with a coverage of more than 90%and a similarity of 41.59%compared to the NctB amino acid sequence,was selected as a template for homology modeling.The obtained model was named nctB.pdb.Ramachandran Plot evaluation results show that the protein model allows the region to be greater than 98%,ie the simulation results are good.Then use the autodock vina molecular docking software has sought cofactor FAD and substrate 6HNor in NctB combination.The results show that the cofactor FAD and 6HNor pyrrole ring C7 there is hydrogen bond,and N11 in the NctB model has hydrogen bonds in the N11 of the Glu292 and 6HNor pyrrole rings.That is,NctB converts 6HNor to 6HM by removing the hydrogen from the 6HNor pyrrole ring C7 and N11.This was consistent with the previously identified 6HM structure.