Study Of Molybdenum Cofactor In Nicotine Metabolism Pathway Of Pseudomonas Putida S16

Nicotine is the main toxic alkaloid in the tobacco manufacture,which seriously threatens the ecological environment and human health.Pseudomonas putida S16 can efficiently degrade nicotine and its pyrrolidine metabolism pathway is highly representative. The key enzyme 3-succinylpyridine monooxygenase catalyzes 3-succinylpyridine to 6-hydroxy-3-succinylpyridine, molybdenum cofactor MCD is crucial for its holoenzyme activity. Higher valence molybdenum ion has very good water solubility and was found in the active electron transfer sites of several oxidoreductases. 3-Succinylpyridine monooxygenase belongs to xanthine dehydrogenase family（XdhABC）. Such oxidases exist in N-heterocyclic compounds biodegradation pathways, including nicotinate,isonicotinate and nicotine. The study of molybdenum cofactor in the pyrrolidine pathway contributes to a better understanding of the molecular mechanism of nicotine metabolism, and lays the foundation for the enzymatic properties and structural information of 3-succinylpyridine monooxygenase.Based on the fact that the heterologous expression of3-succinylpyridine monooxygenase has no activity, we select and disrupt several possible genes related to molybdenum cofactor biosynthesis by digging Pseudomonas putida S16 genomic data. The result indicates that pps₄397 plays an important role in the molybdenum cofactor biosynthetic pathway and it is identified as moaE.RT-qPCR analysis reveals the transcriptional levels of moaE, spmA, and spmC of P. putida S16 in nicotine medium are distinctly higher than those in the glycerol medium. The mutant strain P. putida S16 dmoaE can not use nicotine as the sole carbon and nitrogen source and the gene complemented strain regains the growth ability. Resting cell reaction of S16 dmoaE accumulates 3-succinylpyridine.This paper also conducts a preliminary study of molybdate transporter gene in Pseudomonas putida S16. The genes pps₃310,pps₃311 and pps₃312 probably code molybdate transporter proteins ModABC. ICP-MS results show that tungstate may compete substrate binding sites of ModABC. Resting cell reaction in different culture condition implies that Pseudomonas putida S16 exists additional molybdate transport pathway which is metabolic coercive.