Detection of a C4a-Hydroperoxyflavin Intermediate in the Reaction of a Flavoprotein Oxidase

Sucharitakul, Jeerus; Prongjit, Methinee; Haltrich, Dietmar; Chaiyen, Pimchai

doi:10.1021/bi801039d

Cited by 88 publications

(158 citation statements)

References 35 publications

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“…For oxidases, it is not clear whether the C4a-(hydro)peroxyflavin is formed during the catalytic cycle. Recently, for the first time, such an intermediate has been experimentally detected in a flavoprotein oxidase: pyranose 2-oxidase (34). The oxygenated flavin species decayed to form oxidized flavin and hydrogen peroxide, consistent with the inability of pyranose 2-oxidase to perform oxygenation reactions.…”

Section: Discussionmentioning

confidence: 82%

Identification of a Gatekeeper Residue That Prevents Dehydrogenases from Acting as Oxidases

Leferink

Fraaije

Joosten

et al. 2009

Journal of Biological Chemistry

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The oxygen reactivity of flavoproteins is poorly understood. Here we show that a single Ala to Gly substitution in L-galactono-␥-lactone dehydrogenase (GALDH) turns the enzyme into a catalytically competent oxidase. GALDH is an aldonolactone oxidoreductase with a vanillyl-alcohol oxidase (VAO) fold. We found that nearly all oxidases in the VAO family contain either a Gly or a Pro at a structurally conserved position near the C4a locus of the isoalloxazine moiety of the flavin, whereas dehydrogenases prefer another residue at this position. Mutation of the corresponding residue in GALDH (Ala-113 3 Gly) resulted in a striking 400-fold increase in oxygen reactivity, whereas the cytochrome c reductase activity is retained. The activity of the A113G variant shows a linear dependence on oxygen concentration (k ox ‫؍‬ 3.5 ؋ 10 5 M ؊1 s ؊1 ), similar to most other flavoprotein oxidases. The Ala-113 3 Gly replacement does not change the reduction potential of the flavin but creates space for molecular oxygen to react with the reduced flavin. In the wild-type enzyme, Ala-113 acts as a gatekeeper, preventing oxygen from accessing the isoalloxazine nucleus. The presence of such an oxygen access gate seems to be a key factor for the prevention of oxidase activity within the VAO family and is absent in members that act as oxidases.The flavoenzyme L-galactono-␥-lactone dehydrogenase (GALDH; 2 EC 1.3.2.3) catalyzes the terminal step in the biosynthesis of vitamin C (L-ascorbate) in plants. Besides producing this essential nutrient, GALDH is required for the accumulation of plant respiratory complex I (1). GALDH is an aldonolactone oxidoreductase that belongs to the vanillyl-alcohol oxidase (VAO) flavoprotein family (2). Members of this family share a two-domain folding topology with a conserved FAD binding domain and a cap domain that defines the substrate specificity (3). VAO family members include enzymes involved in carbohydrate metabolism and lignin degradation and enzymes that participate in the synthesis of antibiotics and alkaloids (4). Most VAO members contain a covalently tethered FAD and act as oxidases that use molecular oxygen to reoxidize the flavin, resulting in the production of hydrogen peroxide. In contrast to related aldonolactone oxidoreductases like L-gulono-␥-lactone oxidase from animals (5) and D-arabinono-␥-lactone oxidase from yeast (6), GALDH reacts poorly with molecular oxygen and contains non-covalently bound FAD (7). No crystal structure is available for the aldonolactone oxidoreductase subfamily, and little is known about the nature of the active site and the catalytic mechanism.GALDH is localized in the mitochondrial intermembrane space, where it feeds electrons into the respiratory chain. Its subcellular localization could provide a rationale why GALDH is a dehydrogenase and not, like related enzymes, an oxidase. The latter activity would result in high levels of mitochondrial hydrogen peroxide that promote GALDH inactivation (8) and induce aging, senescence, and cell death (9, 10). Furthermore, i...

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Section: Discussionmentioning

confidence: 82%

Identification of a Gatekeeper Residue That Prevents Dehydrogenases from Acting as Oxidases

Leferink

Fraaije

Joosten

et al. 2009

Journal of Biological Chemistry

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“…Therefore, k obs of both phases in Figure 1 can be accurately estimated; k obs , however, cannot be simply equated with a specific rate constant of any particular step as illustrated in refs (13,22). Since observed rate constants (k obs ) of the first phase (data from A 395 ) are hyperbolically dependent on D-glucose concentrations (Figure 2A), this indicates that the first observed phase is not a direct binding of D-glucose to the enzyme but it is an isomerization of an initial enzyme-substrate complex after the initial binding (21).…”

Section: Resultsmentioning

confidence: 99%

“…Recent investigation on steady-state kinetics of P2O from T. ochracea shows the difference in the kinetic mechanism at pH below and above 7.0 (12). Investigation on the oxidative half-reaction of P2O has shown for the first time the presence of a C4a-hydroperoxy-flavin intermediate in the class of flavoprotein oxidases (13). Similarly, the C4a-adduct intermediate was detected in the crystal structure of choline oxidase (14).…”

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confidence: 99%

Kinetic Mechanism of Pyranose 2-Oxidase from Trametes multicolor

et al. 2009

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Pyranose 2-oxidase (P2O) from Trametes multicolor is a flavoprotein oxidase that catalyzes the oxidation of aldopyranoses by molecular oxygen to yield the corresponding 2-keto-aldoses and hydrogen peroxide. P2O is the first enzyme in the class of flavoprotein oxidases, for which a C4a-hydroperoxy-flavin adenine dinucleotide (FAD) intermediate has been detected during the oxidative half-reaction. In this study, the reduction kinetics of P2O by D-glucose and 2-d-D-glucose at pH 7.0 was investigated using stopped-flow techniques. The results indicate that D-glucose binds to the enzyme with a two-step binding process; the first step is the initial complex formation, while the second step is the isomerization to form an active Michaelis complex (E-Fl ox :G). Interestingly, the complex (E-Fl ox : G) showed greater absorbance at 395 nm than the oxidized enzyme, and the isomerization process showed a significant inverse isotope effect, implying that the C2-H bond of D-glucose is more rigid in the E-Fl ox :G complex than in the free form. A large normal primary isotope effect (k H /k D = 8.84) was detected in the flavin reduction step. Steady-state kinetics at pH 7.0 shows a series of parallel lines. Kinetics of formation and decay of C-4a-hydroperoxy-FAD is the same in absence and presence of 2-keto-D-glucose, implying that the sugar does not bind to P2O during the oxidative half-reaction. This suggests that the kinetic mechanism of P2O is likely to be the ping-pong-type where the sugar product leaves prior to the oxygen reaction. The movement of the active site loop when oxygen is present is proposed to facilitate the release of the sugar product. Correlation between data from presteady-state and steady-state kinetics has shown that the overall turnover of the reaction is limited by the steps of flavin reduction and decay of C4a-hydroperoxy-FAD.Pyranose 2-oxidase (P2O; 1 pyranose:oxygen 2-oxidoreductase; EC 1.13.10) is a flavoprotein oxidase catalyzing the oxidation of several aldopyranoses by molecular oxygen at the C2 position to yield the corresponding 2-keto-aldoses and hydrogen peroxide (Scheme 1) (1). The enzyme was identified and isolated from several species of fungi (2) and is thought to be involved in lignin degradation by providing H 2 O 2 for lignin peroxidase (3). H 2 O 2 production by P2O can also be important for maintaining an oxidative stress level that helps control the growth of other competing organisms (4). The regiospecific oxidation at the pyranose C2 position catalyzed by P2O is a very useful reaction for carbohydrate syntheses since it can be applied in the syntheses of D-tagatose (5), cortalcerone (6), and other valuable sugar synthons (2, 4).P2O from Trametes multicolor is a homotetrameric enzyme with a native molecular mass of 270 kDa (subunit molecular mass of 68 kDa) (1). Each subunit contains one flavin adenine dinucleotide (FAD) covalently attached to the N3 of His167 (7). The enzyme sequence and structure indicate that P2O belongs to the glucose-methanol-choline (GMC) oxidored...

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“…Then, the reduced enzyme solution was transferred into a glass tonometer. Solutions of various concentrations of oxygen were prepared by equilibrating buffers with air and certified oxygen-nitrogen gas mixtures (29).…”

Section: Methodsmentioning

confidence: 99%

Interactions with the Substrate Phenolic Group Are Essential for Hydroxylation by the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Tongsook

Sucharitakul

Thotsaporn

et al. 2011

Journal of Biological Chemistry

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plex, which in turn promotes oxygen atom transfer via an electrophilic aromatic substitution mechanism. Analysis of Ser-146 variants revealed that this residue is necessary for but not directly engaged in hydroxylation. Product formation in S146A is pH-independent and constant at ϳ70% over a pH range of 6 -10, whereas product formation for S146C decreased from ϳ65% at pH 6.0 to 27% at pH 10.0. These data indicate that the ionization of Cys-146 in the S146C variant has an adverse effect on hydroxylation, possibly by perturbing formation of the His ␦؉ ⅐HPA ␦؊ complex needed for hydroxylation.Incorporation of single oxygen atoms into organic compounds (monooxygenation) by hydroxylation or epoxidation is an important biological process for aerobic organisms. The monooxygenation reaction is generally catalyzed by cytochrome P450, metalloenzymes, pterin-dependent and flavindependent monooxygenases (1). Flavin-dependent monooxygenases are involved in a wide variety of biological processes (2, 3). These enzymes catalyze the monooxygenation of many aromatic and aliphatic compounds (3).Based on the number of proteins required for catalysis, flavin-dependent monooxygenases can be classified into singleprotein component and two-protein component types (2-5). Besides an organic substrate, both types of enzymes require NAD(P)H and oxygen as co-substrates. The initial part of the reaction is the reduction of an enzyme-bound flavin by NAD(P)H to generate reduced flavin followed by the reaction of reduced flavin with oxygen to form the C4a-(hydro)peroxy flavin, a key intermediate that is required for oxygenation of an organic substrate. Oxygenation occurs via an oxygen atom transfer from C4a-hydroperoxy flavin to an organic substrate, resulting in the formation of a C4a-hydroxy flavin intermediate and an oxygenated product. The C4a-hydroxy flavin dehydrates at the last step of the reaction to form the final species, oxidized flavin (2-4, 6). All steps for the reactions of singleprotein component flavin-dependent monooxygenases occur within the same polypeptide, whereas for the two-protein component type the flavin reduction occurs on a reductase component and the oxygenation occurs on an oxygenase component (4 -8). The mechanism by which the reduced flavin is transferred involves simple diffusion or protein-protein contacts (5,8). Although single-component flavin-dependent monooxygenases have been studied for more than 40 years, two-component flavin-dependent monooxygenases have only received significant attention during the past decade after recent discoveries of their involvement in a wide variety of reactions (2, 5).The mechanism by which the oxygen atom transfer occurs in flavin-dependent monooxygenases is well understood for only a few enzymatic systems. The best understood oxygenation reaction is the hydroxylation of aromatic compounds catalyzed *

show abstract

Detection of a C4a-Hydroperoxyflavin Intermediate in the Reaction of a Flavoprotein Oxidase

Cited by 88 publications

References 35 publications

Identification of a Gatekeeper Residue That Prevents Dehydrogenases from Acting as Oxidases

Identification of a Gatekeeper Residue That Prevents Dehydrogenases from Acting as Oxidases

Kinetic Mechanism of Pyranose 2-Oxidase from Trametes multicolor

Interactions with the Substrate Phenolic Group Are Essential for Hydroxylation by the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

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