Structural and mechanistic analyses of a nicotine- degrading enzyme from Pseudomonas putida: towards design of tools and biotherapeutics
Tararina, Margarita Alexandrovna
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Tobacco-soil bacteria have evolved not only to tolerate high concentrations of nicotine, but to degrade it as a primary growth source. The genomes of several of these species have been sequenced, allowing for the identification of unique bacterial degradation pathways. In the Gram-negative bacteria, Pseudomonas putida, the nicotine-degrading gene cluster has been described; the encoded enzymes catabolize nicotine via the pyrrolidine pathway, ultimately forming malate and fumarate. In previous studies, the flavoenzyme, nicotine oxidoreductase (NicA2), has been identified as the first committed step of nicotine catabolism in this organism. Preliminary kinetic analysis reported that NicA2 has high specificity for S-nicotine, but a slow catalytic rate. Taking advantage of its unique evolutionary adaptation, we aim to refine the inherent catalytic function and structural features of NicA2 towards the development of a biotherapeutic for nicotine addiction, nicotine poisoning and tools for nicotine biosensor development. Our goal is to identify the factors contributing to the mechanistic and substrate-binding properties of NicA2 to improve its biotherapeutic potential. This work presents the first crystal structure of NicA2, resolved to 2.2 Å resolution, establishing it as a member of the flavin-dependent amine oxidase family with a conserved amine oxidase fold. Structural analysis identified a unique composition of the canonical aromatic cage (W427 and N462), which flanks the flavin isoalloxazine ring. Additionally, the X-ray crystallographic structure of the NicA2/S-nicotine complex was refined to 2.6 Å resolution, revealing a hydrophobic active site in support of a hydride-transfer mechanism. Analysis of enzyme activity with a series of substrate analogs and kinetic analysis of active-site residues reveal the determinants of substrate binding affording the remarkable specificity of this enzyme. Using site-directed mutagenesis of aromatic cage residues, along with analysis of the kinetics of the reductive and oxidative steps, we demonstrate that the rate-limiting reaction step is in the oxidative half-reaction. Structural analysis of an active-site variant revealed a secondary binding site consistent with kinetic analysis demonstrating substrate inhibition. Together, our findings provide kinetic and structural evidence for the catalytic mechanism of NicA2, expanding the possibilities for the generation of catalytically-efficient variants and supporting its role as a promising therapeutic strategy.