Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues

Abstract

The flavoenzyme L-6-hydroxynicotine oxidase (LHNO) is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of carbon-carbon bond, it has recently been shown to catalyze oxidation of a carbon-nitrogen bond (Fitzpatrick et al., Biochemistry 55, 697–703). The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and the roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with the flavin N(5). The N166A and Y311F mutations result in decreases of ~30 and ~4-fold in the kcat/Km and kred values for (S)-6-hydroxynicotine, respectively, with larger effects on the kcat/Km value for (S)-6-hydroxynornicotine. The K287M mutation results in a decrease of ~10-fold in these parameters and a 6,000-fold in the kcat/Km value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the kcat/Km value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to hydride transfer to the flavin.

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase

“…NicA2’s poor reactivity with O 2 is also difficult to rationalize based on structures, as NicA2’s FAD binding site is very similar to that in a homologous protein that reacts rapidly with O 2 , namely 6-hydroxy-L-nicotine oxidase ( Supplementary Fig. 1 ) 29 , 30 . Two recent studies attempted to screen NicA2 variants with residue mutations near the isoalloxazine or the presumed product exit site for improved O 2 -dependent nicotine-degrading activity 12 , 31 .…”

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

“…The average of the two p K a values increases 0.5 unit with ( R )-6-hydroxynornicotine as the substrate. The p K a of the amine nitrogen in this substrate (9.5) is 0.9 unit higher than that of the physiological substrate (8.6), establishing that the p K a of the substrate contributes to the k cat / K amine –pH profile. The pH independence of the k red value with ( R )-6-hydroxynicotine is consistent with productive binding requiring that both the substrate and the enzyme be properly protonated; consequently, the k cat / K amine profile reflects protonations involved in binding rather than catalysis.…”

The enzymes of microbial nicotine metabolism

“…In addition, mutagenesis of His187, Glu300, and Tyr407 established that they are not involved in catalysis. Subsequent mechanistic studies of the reaction using pH and solvent isotope effects established that the reaction catalyzed by LHNO is the same as other flavin amine oxidases, direct hydride transfer from the uncharged amine to the flavin ( Scheme 4 ) [ 19 – 20 ]. Hydrolysis to form 6-hydroxypseudooxynicotine occurs in solution after release of the oxidized amine from the enzyme.…”