6c71 | pdb_00006c71

Nicotine Oxidoreductase in Complex with S-nicotine

spinqualitylabelsall models 6c71, resolution 2.65Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6c71 is a 4 chain structure with sequence from Psep6. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: FAD, NCT Gene: PPS_4081 (PSEP6) Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed 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 A 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 KM, 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. Crystallography Coupled with Kinetic Analysis Provides Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.,Tararina MA, Xue S, Smith LC, Muellers SN, Miranda PO, Janda KD, Allen KN Biochemistry. 2018 Jul 3;57(26):3741-3751. doi: 10.1021/acs.biochem.8b00384. Epub, 2018 Jun 13. PMID:29812904 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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