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. 2019 May 28;58(21):2534-2541.
doi: 10.1021/acs.biochem.9b00297. Epub 2019 May 10.

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

Paul F Fitzpatrick  1 , Vi Dougherty  1 , Bishnu Subedi  1 , Jesus Quilantan  1 , Cynthia S Hinck  1 , Andreina I Lujan  1 , Jose R Tormos  2
Affiliations

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

Paul F Fitzpatrick et al. Biochemistry. .

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 kcat/ Kamine value with either ( R)-6-hydroxynicotine or the slower substrate ( R)-6-hydroxynornicotine. The effect of pH on the rapid-reaction 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 kcat/ Kamine value by ∼20- and ∼220-fold, respectively, and the E352Q substitution decreases this parameter ∼3800-fold. The kcat/ Kamine-pH profile is bell-shaped. The p Ka 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.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Effect of pH on the kcat/Kamine values for (A) wild-type DHNO with (R)-6-hydroxynicotine (▲) or (R)-6-hydroxynornicotine (●) as the substrate and (B) K348M (▲), E350Q (■), E350A (□), and E352Q (●) DHNO with (R)-6-hydroxynicotine as the substrate. The lines are from fits to eq 3.
Figure 2.
Figure 2.
Hydrogen bond interactions in the active site of DHNO (from Protein Data Bank entry 2BVF). Both orientations of Glu350 are shown.
Scheme 1.
Scheme 1.
Proposed Mechanism for DHNO
Scheme 2.
Scheme 2.
Alternate Substrates for DHNO
Scheme 3
Scheme 3
Scheme 4
Scheme 4
See this image and copyright information in PMC

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