7khn

NicA2 variant N462Y/W427Y in complex with (S)-nicotine

spinqualitylabelsall models 7khn, resolution 2.31Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 7khn is a 1 chain structure with sequence from Pseudomonas putida S16. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.31Å Ligands: FAD, NCT Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function F8G0P2_PSEP6 Publication Abstract from PubMed In Pseudomonas putida, the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of (S)-nicotine to N-methyl-myosmine, which is nonenzymatically hydrolyzed to pseudooxynicotine. Structural analysis reveals a monoamine oxidase (MAO)-like fold with a conserved FAD-binding domain and variable substrate-binding domain. The flavoenzyme has a unique variation of the classic aromatic cage with flanking residue pair W427/N462. Previous mechanistic studies using O2 as the oxidizing substrate show that NicA2 has a low apparent Km of 114 nM for (S)-nicotine with a very low apparent turnover number (kcat of 0.006 s(-1)). Herein, the mechanism of NicA2 was analyzed by transient kinetics. Single-site variants of W427 and N462 were used to probe the roles of these residues. Although several variants had moderately higher oxidase activity (7-12-fold), their reductive half-reactions using (S)-nicotine were generally significantly slower than that of wild-type NicA2. Notably, the reductive half-reaction of wild-type NicA2 is 5 orders of magnitude faster than the oxidative half-reaction with an apparent pseudo-first-order rate constant for the reaction of oxygen similar to kcat. X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with (S)-nicotine (at 2.7 and 2.3 A resolution, respectively) revealed no significant active-site rearrangements. A second substrate-binding site was identified in N462Y/W427Y, consistent with observed substrate inhibition. Together, these findings elucidate the mechanism of a flavoenzyme that preferentially oxidizes tertiary amines with an efficient reductive half-reaction and a very slow oxidative half-reaction when O2 is the oxidizing substrate, suggesting that the true oxidizing agent is unknown. Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2.,Tararina MA, Dam KK, Dhingra M, Janda KD, Palfey BA, Allen KN Biochemistry. 2021 Feb 2;60(4):259-273. doi: 10.1021/acs.biochem.0c00855. Epub, 2021 Jan 19. PMID:33464876 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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