Mechanism of the Flavoprotein d-6-Hydroxynicotine Oxidase: Substrate Specificity, pH and Solvent Isotope Effects, and Roles of Key Active-Site Residues

Abstract

The flavoprotein D-6-hydroxynicotine oxidase catalyzes an early step in the oxidation of (R)nicotine, the oxidation of a carbon-nitrogen bond in the pyrrolidine ring of (R)-6-hydroxynicotine. The enzyme is a member of the vanillyl alcohol oxidase/p-cresol methylhydroxylase family of flavoproteins. The effects of substrate modifications on the steady-state and rapid-reaction kinetic parameters are not consistent with the quinone-methide mechanism of p-cresol methylhydroxylase. There is no solvent isotope effect on the k cat /K amine value with either (R)-6hydroxynicotine or the slower substrate (R)-6-hydroxynornicotine. The effect of pH on the rapidreaction kinetic parameters establishes that only the neutral form of the substrate and the correctly protonated form of the enzyme bind. The active-site residues Lys348, Glu350, and Glu352 are all properly positioned for substrate binding. The K348M substitution has only a small effect on the kinetic parameters; the E350A and E350Q substitutions decrease the k cat /K amine value by ~20-and ~220-fold, respectively, and the E352Q substitution decreases this parameter ~3800-fold. The k cat /K amine-pH profile is bell-shaped. The pK a values in that profile are altered by replacement of (R)-6-hydroxynicotine with (R)-6-hydroxynornicotine as the substrate and by the substitutions for Glu350 and Glu352, although the profiles remain bell-shaped. The results are consistent with a network of hydrogen-bonded residues in the active site being involved in binding the neutral form of the amine substrate, followed by the transfer of a hydride from the amine to the flavin.

Structural Insights into the Substrate Range of a Bacterial Monoamine Oxidase

“…It should also be noted that substrate inhibition has been observed with other members of the FAO family, including P. putida NicA2, Calloselasma rhodostoma LAAO, and Arthrobacter nicotinovorans S-6-hydroxynicotine oxidase. ,− As can be seen in Figure D, we also observe a decrease in the rate of the reaction at extended time points, an effect that occurs more rapidly in the reaction tested at pH 8.5 (blue) than the reaction at pH 7.0 (orange). This may be due to the buildup of peroxide in the Amplex Ultra red assay.…”

“…FTMap identified a secondary hotspot within the tunnel that leads to the active site (Figure A). Substrate inhibition has been observed in kinetic studies with other members of the FAO family ,− and a similar substrate inhibition site was identified in A. nicotinovorans 6-hydroxy-L-nicotine oxidase and in P.…”