Discovery, Characterization, and Kinetic Analysis of an Alditol Oxidase from Streptomyces coelicolor

Heuts, Dominic P. H. M.; Hellemond, Erik W. van; Janssen, Dick B.; Fraaije, Marco W.

doi:10.1074/jbc.m610849200

Cited by 75 publications

(127 citation statements)

References 25 publications

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“…In dimethylglycine oxidase from Arthrobacter globiformis, binding of the product decreases a bimolecular rate constant of the flavin oxidation from 3.42 Â 10 5 to 2.01 Â 10 5 M -1 s -1 (31). In the case of alditol oxidase from Streptomyces coelicolor, the bimolecular rate constant for (32). However, there is no direct evidence confirming that the oxidized D-xylose was still bound when the reduced enzyme reacted with oxygen during the stopped-flow experiments.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

confidence: 99%

Kinetic Mechanism of Pyranose 2-Oxidase from Trametes multicolor

et al. 2009

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“…For AldO, where no C4a-(hydro)peroxyflavin intermediate is detected during flavin reoxidation (35), it was argued that the methyl side chain of Ala-105, homol- ogous to Ala-113 in GALDH, could hamper the formation of the peroxy-flavin intermediate by steric hindrance (14). AldO and GALDH show 25% sequence identity, but a high degree of conservation between these enzymes is observed in the area around the isoalloxazine ring of the flavin, including several residues in the substrate binding pocket.…”

Section: Discussionmentioning

confidence: 99%

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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“…In the coupled H 2 O 2 detection assay, horseradish peroxidase (HRP [Sigma], 20 or 50 U/ml) oxidizes 4-aminoantipyrine (0.1 mM) and 3,5-dichloro-2-hydroxybenzenesulfonic acid (1 mM) to form a pink product that was measured at 515 nm (ε 515 ϭ 26 mM Ϫ1 cm Ϫ1 ) (18). Oxygen concentrations were determined with REDFLASH sensor spots and a Firesting O 2 detector and light source (Pyroscience, Aachen, Germany).…”

Section: Methodsmentioning

confidence: 99%

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

Dijkman

Fraaije

2014

Appl Environ Microbiol

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In the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein from Methylovorus sp. strain MP688 in Escherichia coli and subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.

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Discovery, Characterization, and Kinetic Analysis of an Alditol Oxidase from Streptomyces coelicolor

Cited by 75 publications

References 25 publications

Kinetic Mechanism of Pyranose 2-Oxidase from Trametes multicolor

Kinetic Mechanism of Pyranose 2-Oxidase from Trametes multicolor

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

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

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