Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme

• Published in: Biochemistry (Easton) 2018-07, Vol.57 (26), p.3741-3751
• Main Authors: Tararina, Margarita A, Xue, Song, Smith, Lauren C, Muellers, Samantha N, Miranda, Pedro O, Janda, Kim D, Allen, Karen N
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding Sites; Catalytic Domain; Crystallography, X-Ray; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Kinetics; Models, Molecular; Monoamine Oxidase - chemistry; Monoamine Oxidase - metabolism; Nicotine - chemistry; Nicotine - metabolism; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Pseudomonas putida - chemistry; Pseudomonas putida - enzymology; Pseudomonas putida - metabolism; Substrate Specificity
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.8b00384

Nicotine oxidoreductase (NicA2) is a bacterial flavoenzyme, which catalyzes the first step of nicotine catabolism by oxidizing S-nicotine into N-methyl-myosmine. It has been proposed as a biotherapeutic for nicotine addiction because of its nanomolar substrate binding affinity. The first crystal structure of NicA2 has been reported, establishing NicA2 as a member of the monoamine oxidase (MAO) family. However, substrate specificity and structural determinants of substrate binding and/or catalysis have not been explored. Herein, analysis of the pH–rate profile, single-turnover kinetics, and binding data establish that pH does not significantly affect the catalytic rate and product release is not rate-limiting. The X-ray crystal structure of NicA2 with S-nicotine refined to 2.65 Å resolution reveals a hydrophobic binding site with a solvent exclusive cavity. Hydrophobic interactions predominantly orient the substrate, promoting the binding of a deprotonated species and supporting a hydride-transfer mechanism. Notably, NicA2 showed no activity against neurotransmitters oxidized by the two isoforms of human MAO. To further probe the substrate range of NicA2, enzyme activity was evaluated using a series of substrate analogues, indicating that S-nicotine is the optimal substrate and substitutions within the pyridyl ring abolish NicA2 activity. Moreover, mutagenesis and kinetic analysis of active-site residues reveal that removal of a hydrogen bond between the pyridyl ring of S-nicotine and the hydroxyl group of T381 has a 10-fold effect on K M, supporting the role of this bond in positioning the catalytically competent form of the substrate. Together, crystallography combined with kinetic analysis provides a deeper understanding of this enzyme’s remarkable specificity.