Structural And Functional Basis For The Key Protein NicA2 And NicR2 In Nicotine Degradation From Pseudomonas

Nicotine is an environmental toxicant in tobacco wastes,imposing severe hazards for the health of human.Pseudomonas putida S16 is one of the most well studied bacteria capable of efficiently degrading nicotine via the pyrrolidine pathway,and utilizing its by-products as sole carbon and nitrogen sources for growth.Based on the foundation of former research,this work has made a systematic investigation on the structure and function of the first key enzyme NicA2,and the regulator NicR2 in the nicotine degradation pathway.In the nicotine degradation pathway of Pseudomonas putida S16,the pyrrolidine ring of nicotine is dehydrogenated by the nicotine oxidoreductases NicA2 which acts as the first key enzyme in the pathway.In this study,we determined the crystal structure of NicA2 from P.putida S16 in complex with its cofactor FAD,as well as the ternary complex of NicA2 with FAD and the substrate nicotine.Unlike other related structures,NicA2 does not have an associated lipid,but wraps around nicotine more tightly.Notably,our structure reveals that the nicotine substrate is completely buried inside the active site pocket of NicA2 and many bulky residues occlude the exit passage of pseudooxynicotine(PN).Mutation of bulky residues at the PN-exit tunnel to slimmer ones enhanced the catalytic turnover rate and induced the accumulation of PN,but hindered bacterial growth.Normal growth could be rescued by coexpression of the downstream Pnao enzyme that decomposes PN.Therefore,the occluded tunnel of NicA2 serves as a “molecular decelerating” which slows down the release of the toxic product PN through its exit,which prevents PN from accumulating and causing damage to the bacterium.NicA2 belongs to amine oxidases.Flavin-dependent amine oxidases have received extensive attention on their importance in drug metabolisms,Parkinson's disease and neurological disorders.In this study,we find NicA2 shares similarity to MAOs and both of them formed the “aromatic cave” to deeply bury their substrates.Alignments with other amine oxidases revealed that these enzymes,including human MAOA andMAOB,also possess residues with bulky side-chains in the exit passage,besides,mutation of residues on the product-exit passage of MAOs increase its catalytic turnover rate,implying that similar product sequestration mechanisms might also exist for these enzymes.Since severe depression and Parkinson's disease could be led due to the excessive catalytic product of MAOs,it is speculated that MAOs may launch the “molecular decelerating” mechanism to precisely regulate the neurotransmitters.NicR2,a member in TetR-like family,acts as the transcriptional regulator that regulates nic2 cluster in the nicotine degradation pathway from Pseudomonas putida S16.We solved the crystal structures of NicR2 and its complex with the inducer6-hydroxy-3-succinoyl-pyridine(HSP).NicR2 is composed of a DNA-binding domain,and a ligand-binding domain.Residues R91,Y114 and Q118 of NicR2 form hydrogen bonds with HSP,their indispensable roles in NicR2's recognition with HSP were confirmed.Further analysis on the active pocket indicated that NicR2 selectively recognize HSP,but not other intermediates,as its specific inducer.We made a careful comparison of the amino acid sequence and the crystal structure of the DBD domain of NicR2 with those of QacR and SimR.Six residues were surmised as putative DNA-binding residues and we further identified the residues directly participating in DNA-binding.Interestingly,both NicR2-DBD and N-terminal extension are required for the association with the operator DNA.Finally,we proposed that either NicR2 or the DNA would undergo a conformational change upon their association.Altogether,our structural and biochemical investigations unravel how NicR2 selectively recognizes HSP and DNA,and provide new insights into the TetR family of repressors.The systematic work about the structure and function of the key protein has shed considerable light on the molecular mechanism of nicotine degradation and lays a solid theoretical and scientific foundation for orientational alteration for the key enzymes.