Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

Fitzpatrick, Paul F.

doi:10.1002/jlcr.1400

Cited by 23 publications

(22 citation statements)

References 69 publications

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“…Here, we investigated the reaction mechanism of plant GOX by taking advantage of 12 C/ 13 C and hydrogen/deuterium isotope effects (Table 2 and 3). Because of the conserved structure and residues involved in the active site of plant (6,12,22) and mammalian (9) GOX enzymes, our conclusions concerning the catalytic mechanism (see below) are probably true also for the closely related mammalian enzyme.…”

Section: Atgox1mentioning

confidence: 78%

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

Dellero

Mauve

Boex-Fontvieille

et al. 2015

Journal of Biological Chemistry

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Background: Uncertainty remains about the nature of transition states along the reductive half-reaction of glycolate oxidase. Results: Deuterated glycolate and solvent slow down plant glycolate oxidase catalysis to a modest extent. Conclusion: Isotope effects support a hydride transfer mechanism and indicate glycolate deprotonation to be only partially rate-limiting. Significance: Understanding the catalytic mechanism of the enzyme is crucial for designing drugs/herbicides to inhibit its activity.

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Section: Atgox1mentioning

confidence: 78%

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

Dellero

Mauve

Boex-Fontvieille

et al. 2015

Journal of Biological Chemistry

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“…Various mechanisms have been proposed for the oxidation of amines by flavoproteins [19, 20]. However, the mechanism of L- proline oxidation by ProDH is different in two significant ways: oxidation occurs at a secondary amine and oxidation involves the N1-C5 bond rather than the typical N1-Cα bond.…”

Section: Resultsmentioning

confidence: 99%

Kinetic and Isotopic Characterization of l-Proline Dehydrogenase from Mycobacterium tuberculosis

Serrano

Blanchard

2013

Biochemistry

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The monofunctional proline dehydrogenase (ProDH) from Mycobacterium tuberculosis performs the flavin-dependent oxidation of L-proline to Δ1-pyrroline-5-carboxylate (P5C) in the proline catabolic pathway. The ProDH gene, prub, was cloned into the pYUB1062 vector, and the C-terminal His-tagged 37 kDa protein was expressed and purified by nickel-affinity chromatography. A steady-state kinetic analysis revealed a ping-pong mechanism with an overall kcat of 33 ± 2 sec−1 and Km values of 5.7 ± 0.8 mM and 3.4 ± 0.3 μM for L-proline and 2,6-dichlorophenolindophenol (DCPIP), respectively. The pH dependence of kcat revealed that one enzyme group exhibiting a pK value of 6.8 must be deprotonated for optimal catalytic activity. Site-directed mutagenesis suggests that this group is Lys110. The primary kinetic isotope effects on V/KPro and V of 5.5 and 1.1, respectively, suggest that hydride transfer from L-proline to FAD is rate-limiting for the reductive half-reaction, but that FAD reoxidation is the rate-limiting step in the overall reaction. Solvent and multiple kinetic isotope effects suggest that L-proline oxidation occurs in a stepwise rather than concerted mechanism. Pre-steady state kinetics reveal an overall kred of 88.5 ± 0.7 sec−1, and this rate is subject to a primary kinetic isotope effect of 5.2. These data confirm that the overall reaction is limited by reduced flavin reoxidation in the second half-reaction.

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“…Their catalytic cycle consists of two half reactions. During the reductive half reaction two electrons are transferred from the substrate to the flavin cofactor, a process which is generally believed to occur through the direct transfer of a hydride anion to the N5 atom of the flavin 1. 2 In the oxidative half reaction the flavin cofactor is reoxidized by molecular oxygen, which is reduced to hydrogen peroxide.…”

Section: Steady‐state Kinetic Parameters For the Oxidation Of Thiols mentioning

confidence: 99%

The Oxidation of Thiols by Flavoprotein Oxidases: a Biocatalytic Route to Reactive Thiocarbonyls

Ewing¹,

Dijkman

Vervoort³

et al. 2014

Angewandte Chemie

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Flavoprotein oxidases are a diverse class of biocatalysts, most of which catalyze the oxidation of CÀO, CÀN, or CÀC bonds. Flavoprotein oxidases that are known to catalyze the oxidation of CÀS bonds are rare, being limited to enzymes that catalyze the oxidative cleavage of thioethers. Herein, we report that various flavoprotein oxidases, previously thought to solely act on alcohols, also catalyze the oxidation of thiols to thiocarbonyls. These results highlight the versatility of enzymatic catalysis and provide a potential biocatalytic route to reactive thiocarbonyl compounds, which have a variety of applications in synthetic organic chemistry.Flavoprotein oxidases constitute a diverse class of oxidoreductases, which contain a flavin cofactor in the form of flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). They catalyze the oxidation of a wide range of substrates, with molecular oxygen functioning as the final oxidant. Their catalytic cycle consists of two half reactions. During the reductive half reaction two electrons are transferred from the substrate to the flavin cofactor, a process which is generally believed to occur through the direct transfer of a hydride anion to the N5 atom of the flavin. [1,2] In the oxidative half reaction the flavin cofactor is reoxidized by molecular oxygen, which is reduced to hydrogen peroxide. Unlike many other oxidative enzymes, for example dehydrogenases, oxidases do not require stoichiometric amounts of expensive organic cofactors, making them interesting candidates for biocatalytic exploitation. Most of the characterized flavoprotein oxidases catalyze the oxidation of CÀO, CÀN, or CÀC bonds to the corresponding double bonds.[3] A small number of flavoprotein oxidases are known to oxidize sulfurcontaining compounds. Prenylcysteine lyase (Enzyme Commission (EC) number 1.8.3.5) and farnesylcysteine lyase (EC 1.8.3.6) catalyze the oxidative cleavage of thioethers into aldehydes and thiols, whereas sulfhydryl oxidases (EC 1.8.3.2 and 1.8.3.3) catalyze the formation of disulfide bonds from thiols. So far, no flavoprotein oxidases have been identified that catalyze the oxidation of thiols to thiocarbonyl compounds. Herein, we present the finding that a number of flavin-dependent oxidases, that hitherto had been thought to act solely on alcohols, also oxidize thiols that are structurally similar to their alcohol substrates, yielding the corresponding thiocarbonyls.Alditol oxidase from Streptomyces coelicolor (AldO; EC 1.1.3.41) is a flavin-dependent oxidase that catalyzes the regioselective oxidation of the primary hydroxy groups of a broad range of polyols, with the best substrates being alditols, such as xylitol, d-sorbitol, and l-threitol.[4, 5] AldO displays stereoselectivity: for example, d-sorbitol is converted into its oxidized form significantly more efficiently than its C2 epimer d-mannitol. AldO belongs to the vanillyl alcohol oxidase (VAO) family of structurally homologous flavoproteins. These flavoproteins contain a conserved FAD-binding domain ...

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Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

Cited by 23 publications

References 69 publications

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

Kinetic and Isotopic Characterization of l-Proline Dehydrogenase from Mycobacterium tuberculosis

The Oxidation of Thiols by Flavoprotein Oxidases: a Biocatalytic Route to Reactive Thiocarbonyls

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