The enzymes of microbial nicotine metabolism

Fitzpatrick, Paul F.

doi:10.3762/bjoc.14.204

Cited by 15 publications

(13 citation statements)

References 78 publications

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“…It was isolated from Pseudomonas putida S16, an unusual microorganism that can achieve robust growth using nicotine as its sole carbon and nitrogen source 7 . NicA2 catalyzes the first step in the catabolic pathway, which eventually results in the production of fumarate for primary metabolism 8 . NicA2’s FAD cofactor accepts a hydride from nicotine, converting it into N-methylmyosmine in the enzyme’s biologically important reductive half-reaction 9 .…”

Section: Introductionmentioning

confidence: 99%

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase

et al. 2021

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Nicotine oxidoreductase (NicA2), a member of the flavin-containing amine oxidase family, is of medical relevance as it shows potential as a therapeutic to aid cessation of smoking due to its ability to oxidize nicotine into a non-psychoactive metabolite. However, the use of NicA2 in this capacity is stymied by its dismal O 2 -dependent activity. Unlike other enzymes in the amine oxidase family, NicA2 reacts very slowly with O 2 , severely limiting its nicotine-degrading activity. Instead of using O 2 as an oxidant, we discovered that NicA2 donates electrons to a cytochrome c, which means that NicA2 is actually a dehydrogenase. This is surprising, as enzymes of the flavin-containing amine oxidase family were invariably thought to use O 2 as an electron acceptor. Our findings establish new perspectives for engineering this potentially useful therapeutic and prompt a reconsideration of the term “oxidase” in referring to members of the flavin-containing amine “oxidase” family.

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Section: Introductionmentioning

confidence: 99%

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase

et al. 2021

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“…M icro-organisms derived from tobacco soil have evolved to degrade nicotine as a growth source using two major pathways, the pyridine and the pyrrolidine pathways. 1 In Pseudonomas putida S16, nicotine catabolism occurs via the pyrrolidine pathway using enzymes located on a single gene cluster. 2 The enzyme responsible for the first committed step has been identified as nicotine oxidoreductase (NicA2).…”

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confidence: 99%

“…1 22 with one molecule in the asymmetric unit and the following unit cell dimensions: a = b h i b i t e d .…”

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Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2

et al. 2021

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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 O 2 as the oxidizing substrate show that NicA2 has a low apparent K m of 114 nM for (S)-nicotine with a very low apparent turnover number (k cat 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 k cat . X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with (S)-nicotine (at 2.7 and 2.3 Å resolution, respectively) revealed no significant active-site rearrangements. A second substratebinding 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 O 2 is the oxidizing substrate, suggesting that the true oxidizing agent is unknown.

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“…T here are two major microbial pathways for degradation of the plant alkaloid nicotine. 1,2 In the pathway best characterized in Pseudomonas putida, 3 the initial step is the oxidation of the pyrrolidine ring of (S)-nicotine to form Nmethyl-myosmine catalyzed by the flavoprotein nicotine oxidase. 4,5 In the pathway best characterized in Arthrobacter, 1,6 the initial step is the hydroxylation of the pyridyl ring by the molybdopterin enzyme nicotine dehydrogenase, 7 which is active on both (S)-and (R)-nicotine.…”

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

et al. 2019

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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.

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The enzymes of microbial nicotine metabolism

Cited by 15 publications

References 78 publications

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase

Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2

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

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