| Nicotine is an alkaloid synthesized in tobacco and a toxic N-heterocyclic compound.Tobacco processing produces a lot of wastes,which contain high content of nicotine and threaten the environment and human health.The use of green and efficient means to degrade nicotine is the focus of the disposal of these wastes.Nowadays,traditional physical and chemical methods are often used to treat the wastes,which are likely to cause secondary pollution to the environment.Microbial degradation of nicotine has become the main research direction because of its harmlessness and sustainability.The mechanism of microbial degradation of nicotine has been extensively studied in recent years.For example,our laboratory has isolated a strain of Agrobacterium tumefaciens S33,which can grow with nicotine as the sole source of carbon and nitrogen,and use a novel hybrid of the pyridine and pyrrolidine pathways(also called VPP pathway)to degrade nicotine.Its biochemical and molecular mechanisms of nicotine degradation is gradually elucidated through the recent studies in our laboratory.In the hybrid,the degradation process similar to the pyridine pathway is from nicotine through two steps oxidoreductive reactions to 6-hydroxypseudooxynicotine,which is then oxidized by 6-hydroxypseudooxynicotine dehydrogenase(Pno)to 6-hydroxy-3-succinoyl-semialdehyde-pyridine,The degradation process of the aldehyde intermediate is similar to that of pyrrolidine pathway,where 2,5-dihydroxypyridine is formed from 6-hydroxy-3-succinoyl-semialdehyde-pyridine by two-step of oxidation,and then it enters TCA cycle after the ring opening.Although the biochemical mechanism of this hybrid pathway has been well studied,there are still some problems to be solved.For example,the Pno-catalyzed reaction is not fully understood.The reaction starts at a high rate,and then the reaction rate drops rapidly,resulting in the formation of a small amount of product.In this study,it is speculated that an unstable and toxic imine intermediate is produced in the reaction considering that the substrate 6-hydroxypseudooxynicotine has a secondary amine group on its side chain.Interestingly,there is a gene in the nicotine degradation gene cluster that can encode Rid protein(named Rid-NC).Rid is a widely distributed family of small proteins with multiple functions.Some subfamilies of Rid have deaminase activity to eliminate the toxicity of reactive intermediate imine.Therefore,it is proposed that the Rid-NC protein may be related to the incompleteness of the Pno-catalyzed reaction.In addition,the key enzyme and catalytic mechanism for the formation of 6-hydroxy-3-succinoylpyridine from 6-hydroxy-3-succinoyl-semialdehyde-pyridine in the fifth step of the pathway are also unknown.The ald gene in the nicotine degradation gene cluster is speculated to encode an aldehyde dehydrogenase(Ald)responsible for this reaction.Based on these predictions,this study mainly focuses on characterization of biochemical properties and functions of the key enzymes Rid-NC and Ald in the nicotine metabolism pathway of A.tumefaciens S33.Firstly,Rid-NC was heterologously expressed and purified.In the Pno-catalyzed reaction(6-hydroxypseudooxynicotine is dehydrogenated to 6-hydroxy-3-succinoyl-semialdehyde-pyridine),Rid-NC was added to determine the activity change of Pno,and it was found that the addition of Rid-NC could enhance the Pno-catalyzed reaction.Using pseudooxynicotine instead of 6-hydroxypseudooxynicotine(which has been proved to be replaceable),it was found that Rid-NC could still enhance the catalytic reaction of Pno,suggesting that Rid-NC can reduce the toxicity of imine intermediate producted in the Pno-catalyzed reaction.The substrate and product of the reaction were quantified by HPLC,and it was found that the amount of reaction product increased by at least 5 times after adding Rid-NC.In addtion,the disruption of the rid-NC gene caused strain S33 to grow slowly on nicotine.These evidences indicate that Rid-NC plays an important role in the degradation of nicotine by A.tumefaciens S33.Further analysis shows Rid-NC belongs to the Rid6 subfamily,and there is a second Rid6 subfamily protein named Rid-AA exists in A.tumefaciens S33.The functions of the typical RidA,Rid-AA,and Rid-NC were studied,and it was found that RidA and Rid-AA have the same functions as Rid-NC.Through mutating the conservative amino acid sites of Rid-NC,the reaction mechanism of the imine intermediate hydrolysis in the Pno-catalyzed reaction by Rid-NC was further explored.The process of 6-hydroxypseudooxynicotine dehydrogenation catalyzed by Pno to 6-hydroxy-3-succinoyl-semialdehyde-pyridine was thus clearly explained.After the heterologous expression and purification,Ald was identified through LC-MS analysis to catalyze 6-hydroxy-3-succinoyl-semialdehyde-pyridine into 6-hydroxy-3-succinoyl-pyridine using NAD+ as a coenzyme.The feasibility of using 3-succinoyl-semialdehyde-pyridine instead of 6-hydroxy-3-succinoyl-semialdehyde-pyridine in the Ald-catalyzed reaction was also confirmed.The diruption of the ald gene caused strain S33 to grow slowly on nicotine,indicating that Ald plays an important role in the nicotine degradation of A.tumefaciens S33.The optimal pH value and the apparent Km value of Ald-catalyzed reaction with different substrates including 3-succinoyl-semialdehyde-pyridine,benzaldehyde,furfural,and acetaldehyde were also determined.Considering the value of furan compounds in different fields,the conversion efficiency of BL21-Ald recombinant cells was tested to catalyze furfural to furoic acid.The substrate was completely converted after 2 h,and the product conversion rate reached 99%after 15 h.It was verified that Ald can be used in the prodction of bio-based furan chemicals.In this study,it is found that there may be toxic imine intermediates produced in the Pno-catalyzed reaction that affect the Pno activity,and Rid-NC can emilinate the effect through its deaminase activity.The reaction mechanism of Rid-NC on hydrolysis of the toxic imine intermediates was also proposed.The findings further deepen our understanding of the metabolism of toxic N-heterocyclic aromatic compounds in microorganisms.It proves that in addition to amino acid metabolism,Rid family proteins are also involved in the metabolism of N-heterocyclic aromatic alkaloids.The function of Rid6 subfamily proteins in metabolic damage control system was verified for the first time.The ability of reactive imines to damage non-pyridoxal 5'-phosphate dependent enzymes is reported.This study provides new insights into the function of the Rid family proteins.In addition,the ability of Ald to catalyze 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoyl-pyridine was confirmed with NAD+ as coenzyme,and the substrate spectra of Ald was determined.BL21-Ald recombinant cells can effectively catalyze furfural to furoic acid,which provides a new method for production of bio-based furan chemicals.These studies solve the unknown reaction mechanisms in the hybrid pathway of nicotine degradation in A.tumefaciens S33 and demonstrate the preliminary exploration of the application of Ald. |
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