Structural And Functional Studies Of Nfo, Ami, Pp-Iso In The Nicotine Degradation Pathway Of P. Putida Strain S16

Nicotine exists in the toxic waste produced in the tobacco manufacturing process,and imposes a serious hazard for human health and environment. Microbialdegradation of nicotine has attracted more and more attention because of the highspeed, low cost, and less pollution.The pyrrolidine pathway of nicotine degradation in Pseudomonas putida strainS16has recently been revealed. Nfo (N-formylmaleamate deformylase), Ami(maleamate amidase), Pp-Iso (Pseudomonas putida maleate isomerase) catalyze thelast three steps of nicotine degradation in this pathway. Nfo hydrolyzesN-formylmaleamate (NFM) to maleamate. Ami catalyzes the further hydrolysis ofmaleamate to maleate, which is converted to fumarate by the maleate isomerasePp-Iso. Fumarate then enters the Krebs cycle. Thus, nicotine is degraded toenvironmentally non-toxic compounds.In this research, the crystal structures of selenomethionine-substituted Nfo(S94A)mutant, Ami in complex with its reaction product maleate, and Pp-Iso in complexwith its substrate maleate were determined. A model of the Nfo(S94A)/NFM complexstructure was obtained through molecular docking. Residues on these three enzymeswhich interact with their substrates were mutated, and the enzyme kinetic constants ofboth wild type and mutants were analyzed, through which the molecular mechanismof these enzymatic reactions were revealed.The NFM deformylase Nfo and the maleamate amidase Ami in the nicotinecatabolic pathway of P. putida S16represent two typical subfamilies of amidehydrolases: deformylases and amidases. They catalyze two sequential hydrolysis reactions involving similar substrates: NFM and maleamate. Our assay results showedthat Nfo and Ami specifically recognized their respective substrates and wereincapable of replacing each other. Through the molecular docking analysis of NFM ormaleamate to Nfo as well as Ami, the molecular mechanisms of their substratespecificities were revealed.The maleate isomerase Pp-Iso catalyzes the last step in the nicotine degradationpathway of P. putida S16, the cis-trans isomerization of maleate to fumarate. Ourstructure analysis showed that the maleate was completely wrapped inside the Pp-Isoenzyme. Examination of our structure prompted us to hypothesize that the β2-α2loopand the β6-α7loop have a breathing motion that regulates substrate entry and productdeparture. The molecular dynamics simulation as well as the enzymatic activity assayshowed that these two loops play important roles in substrate entry and productdeparture. Thus, results of our structural, computational, as well as biochemicalassays were all consistent with a model that the β2-α2and β6-α7loops have abreathing motion that allows Pp-Iso to completely enfold the substrate maleate insidefor the isomerization reaction to occur smoothly.Our research combine structural, computational, as well as enzymatic activityassays to elucidate the molecular mechanism of Nfo, Ami and Pp-Iso in thepyrrolidine pathway of nicotine degradation in P. putida strain S16. Our resultsprovide an important theoretical basis and practical significance for future rationalalteration of bacteria strains and key enzymes, and lay solid scientific and practicalfoundations for the improvement of the degradation effiency of nicotine inenvironmental wastes.