Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase (MAO) family

Abstract

FAD-linked oxidases constitute a class of enzymes which catalyze dehydrogenation as a fundamental biochemical reaction, followed by reoxidation of reduced flavin. Here, we present high-resolution crystal structures showing the flavoenzyme 6-hydroxy-l-nicotine oxidase in action. This enzyme was trapped during catalytic degradation of the native substrate in a sequence of discrete reaction states corresponding to the substrate-reduced enzyme, a complex of the enzyme with the intermediate enamine product and formation of the final aminoketone product. The inactive d-stereoisomer binds in mirror symmetry with respect to the catalytic axis, revealing absolute stereospecificity of hydrogen transfer to the flavin. The structural data suggest deprotonation of the substrate when bound at the active site, an overall binary complex mechanism and oxidation by direct hydride transfer. The amine nitrogen has a critical role in the dehydrogenation step and may activate carbocation formation at the α-carbon via delocalization from the lone pair to σ* C(α)-H. Enzymatically assisted hydrolysis of the intermediate product occurs at a remote (P site) cavity. Substrate entry and product exit follow different paths. Structural and kinetic data suggest that substrate can also bind to the reduced enzyme, associated with slower reoxidation as compared to the rate of reoxidation of free enzyme. The results are of general relevance for the mechanisms of flavin amine oxidases.

Fig. 1.

Reaction catalyzed by 6HLNO. (A) Dehydrogenation of the pyrrolidine moiety of 6HLN (S) results in the formation of the intermediate product 6-hydroxy-N-methylmyosmine (P1). Subsequent hydration of the intermediate opens the pyrrole ring to form the final product 6-hydroxy-pseudooxynicotine (P2). (B) Crystal structure of 6HLNO subunit with substrate and product sites. The substrate (teal) is bound at the active site and the intermediate product P1 (orange) at the P site. PHL denotes a noncovalently bound phospholipid. The inset shows the solvent-inaccessible active-site cavity (yellow; volume 195 Å³), the exit (P site) cavity (brown; 290ų) opening toward the protein surface, and the channel (red; 80 Å³) for gated transfer of the intermediate product. Arrows indicate possible paths for entry and exit.

Fig. 2.

Enzymatic reaction states populated during catalytic turnover in 6HLNO crystals. Stereoview of E-S-P1 (gray) showing the substrate 6HLN (yellow) bound at the active site and the intermediate product P1 (orange) in the P-site cavity. The structure of the dithionite-reduced complex E-S (teal) is superimposed.

Fig. 3.

Binding geometry at P site. (A) The structures E-S-P1 (orange), E-S-P2 (green), E-S-I (magenta), E-S (yellow), and E_nat (gray) are superimposed. Ligand binding to the P site is associated with backbone motions of the segment 81–85. The orientation in the P-site cavity is shown for (B) the intermediate P1 (orange), (C) the final product P2 (green), and (D) the inhibitor 6HDN (magenta). Simulated annealed omit (F_o - F_c) maps are contoured at 2.2 σ for P1, 1.8 σ for P2, and 3.0 σ for 6HDN.

Fig. 4.

l- and d-stereoisomers bound at active site. The structures E-S-P1 (yellow) and E-I-P2 (teal) are superimposed. The pyrrolidine rings of 6HLN and 6HDN are related by an approximate mirror symmetry. The α-carbon hydrogens (gray) occupy closely similar locations. The pyrrolidine N⁺CH₃-H group of 6HLN may interact with Tyr407 forming a charge-relay system with His187 and Ser197. The amine nitrogen of 6HLN is located opposite the C4a - C4 = O4 locus. The distance between C_α of 6HLN and the flavin N5 is 3.48 Å (pink broken line).