Molecular Mechanism Of Nicotine Catabolism By Pseudomonas Putida S16

The N-heterocyclic compounds in industial and agricultural wastes, possessing carcinogenic, teratogenic, and mutagenic properties, cause damages on the ecological environment and human health. Nicotine, as an important N-heterocyclic compound, is a toxic hazardous waste. Using microorganisms to remove environmental pollutants such as nicotine is the most feasible method. Pseudomonas putida S16, which exhibits high nicotine-degrading activity, utilizes the pyrrolidine pathway to degrade nicotine. However, the pyrrolidine pathway in Pseudomonas is still incomplete. The catabolic pathway of 2,5-dihydroxy-pyridine(DHP) and the key enzymes that catalyze 3-succinoyl-pyridine(SP) are unknown. Moreover, the regulatory mechanism of nicotine catabolism in Pseudomonas has not been reported.The gene iso which might be responsible for transforming maleic acid to fumaric acid was obtained from strain S16, and then heterologously expressed in Escherichia coli. The encoding product of iso, Pp-Iso, was identified as maleic acid isomerase which is responsible for the last step of pyrrolidine pathway.In the past few years, there have been significant efforts to identify the key gene(s) for the hydroxylation of SP, including genome library screening and wild-type enzyme purification. However, these efforts did not result in identifying any genes related to SP. Here, we identified 3 overlapping genes, spmA, spmB and spmC. By constructing spmABC genes disruption mutant S16 dspm, SpmABC were suggested to be SP hydroxylase that converts SP to 6-hydroxy-3-succinoyl-pyridine(HSP). However, when the spm ABC genes were expressed in E. coli, inactive SP hydroxylase was formed. It may be explained by lacking the molybdenum molybdopterin cytosine dinucleotide cofactor(MCD), since E. coli may be unable to synthesize the MCD. This is the reason why we failed to obtain the spmABC genes using several other methods over the past few years. In addition, active SP hydroxylase was detected in Pseudomonas by constructing spmABC complement mutant.The pathways and biochemical mechanism of nicotine degradation have been documented in detail; however, the regulatory mechanism has not been well studied. Here, we identified the first regulator, NicR2, from Pseudomonas involved in nicotine degradation. NicR2 was suggested to be the repressor of nic2 cluster by constructing nicR2 gene disruption and complement mutants. Electrophoretic mobiliy shift assay(EMSA) and DNase I footprinting indicated that the binding site of NicR2 is a 28 bp inverted repeat(IR) and HSP is regarded as the effector.We further investigated the binding model for NicR2 from P. putida S16 binding to the IR. Isothermal titration calorimetry and biolayer interferoetry suggested that two NicR2 dimers bind to the IR cooperatively. The motif “CTATATGN6~8CATATAA” is important for sequence-specific cooperative DNA binding by Nic R2. Using EMSAs, we found that both NicR2 dimer and tetramer can bind to the half-site of the IR. In additon, we observed NicR2 tetramers, which suggest protein-protein interactions, using superdex 200 and protein cross-linking. Based on the above results, we propose a DNA-binding model: two NicR2 dimers bind to the IR through protein-protein interactions with each dimer binding the half-site of the IR. This is different from the known models of previously characterized DNA-binding proteins that use HTHs for DNA recognition, however, resembles some regulators that use β-sheet for DNA binding. The regulator of nicotine degradation in Pseudomonas highlights a new level of complexity in prokaryotic transcriptional regulation.