6C71

Nicotine Oxidoreductase in Complex with S-nicotine

Summary for 6C71

• Entry DOI: 10.2210/pdb6c71/pdb
• Descriptor: Amine oxidase, (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE, FLAVIN-ADENINE DINUCLEOTIDE, ... (4 entities in total)
• Functional Keywords: flavoprotein, monoamine oxidase family, nicotine, substrate specificity
• Biological source: Pseudomonas putida (strain S16)
• Total number of polymer chains: 4
• Total formula weight: 218452.71

Authors

Tararina, M.A.,Allen, K.N. (deposition date: 2018-01-19, release date: 2018-07-11, Last modification date: 2023-10-04)

Primary citation

Tararina, M.A.,Xue, S.,Smith, L.C.,Muellers, S.N.,Miranda, P.O.,Janda, K.D.,Allen, K.N.
Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.
Biochemistry, 57:3741-3751, 2018

PubMed Abstract: 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, 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.

PubMed: 29812904
DOI: 10.1021/acs.biochem.8b00384

Experimental method

X-RAY DIFFRACTION (2.649 Å)