Publications

• Oxidation mode of pyranose 2-oxidase is controlled by pH.

  Prongjit M., Sucharitakul J., Palfey B.A., Chaiyen P.

  Pyranose 2-oxidase (P2O) from Trametes multicolor is a flavoenzyme that catalyzes the oxidation of d-glucose and other aldopyranose sugars at the C2 position by using O₂ as an electron acceptor to form the corresponding 2-keto-sugars and H₂O₂. In this study, the effects of pH on the oxidative half ... >> More

  Pyranose 2-oxidase (P2O) from Trametes multicolor is a flavoenzyme that catalyzes the oxidation of d-glucose and other aldopyranose sugars at the C2 position by using O₂ as an electron acceptor to form the corresponding 2-keto-sugars and H₂O₂. In this study, the effects of pH on the oxidative half-reaction of P2O were investigated using stopped-flow spectrophotometry. The results showed that flavin oxidation occurred via different pathways depending on the pH of the environment. At pH values lower than 8.0, reduced P2O reacts with O₂ to form a C4a-hydroperoxyflavin intermediate, leading to elimination of H₂O₂. At pH 8.0 and higher, the majority of the reduced P2O reacts with O₂ via a pathway that does not allow detection of the C4a-hydroperoxyflavin, and flavin oxidation occurs with decreased rate constants upon the rise in pH. The switching between the two modes of P2O oxidation is controlled by protonation of a group which has a pK(a) of 7.6 ± 0.1. Oxidation reactions of reduced P2O under rapid pH change as performed by stopped-flow mixing were different from the same reactions performed with enzyme pre-equilibrated at the same specified pH values, implying that the protonation of the group which controls the mode of flavin oxidation cannot be rapidly equilibrated with outside solvent. Using a double-mixing stopped-flow experiment, a rate constant for proton dissociation from the reaction site was determined to be 21.0 ± 0.4 s⁻¹. << Less

  Biochemistry 52:1437-1445(2013) [PubMed] [EuropePMC]

• Kinetic mechanism of pyranose 2-oxidase from trametes multicolor.

  Prongjit M., Sucharitakul J., Wongnate T., Haltrich D., Chaiyen P.

  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- ... >> More

  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 pre-steady-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. << Less

  Biochemistry 48:4170-4180(2009) [PubMed] [EuropePMC]

• The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily.

  Wongnate T., Chaiyen P.

  Enzymes in the glucose-methanol-choline (GMC) oxidoreductase superfamily catalyze the oxidation of an alcohol moiety to the corresponding aldehyde. In this review, the current understanding of the sugar oxidation mechanism in the reaction of pyranose 2-oxidase (P2O) is highlighted and compared wit ... >> More

  Enzymes in the glucose-methanol-choline (GMC) oxidoreductase superfamily catalyze the oxidation of an alcohol moiety to the corresponding aldehyde. In this review, the current understanding of the sugar oxidation mechanism in the reaction of pyranose 2-oxidase (P2O) is highlighted and compared with that of other enzymes in the GMC family for which structural and mechanistic information is available, including glucose oxidase, choline oxidase, cholesterol oxidase, cellobiose dehydrogenase, aryl-alcohol oxidase, and pyridoxine 4-oxidase. Other enzymes in the family that have been newly discovered or for which less information is available are also discussed. A large primary kinetic isotope effect was observed for the flavin reduction when 2-d-D-glucose was used as a substrate, but no solvent kinetic isotope effect was detected for the flavin reduction step. The reaction of P2O is consistent with a hydride transfer mechanism in which there is stepwise formation of d-glucose alkoxide prior to the hydride transfer. Site-directed mutagenesis of P2O and pH-dependence studies indicated that His548 is a catalytic base that facilitates the deprotonation of C2-OH in D-glucose. This finding agrees with the current mechanistic model for aryl-alcohol oxidase, glucose oxidase, cellobiose dehydrogenase, methanol oxidase, and pyridoxine 4-oxidase, but is different from that of cholesterol oxidase and choline oxidase. Although all of the GMC enzymes share similar structural folding and use the hydride transfer mechanism for flavin reduction, they appear to have subtle differences in the fine-tuned details of how they catalyze substrate oxidation. << Less

  FEBS J 280:3009-3027(2013) [PubMed] [EuropePMC]

• Identification of a catalytic base for sugar oxidation in the pyranose 2-oxidase reaction.

  Wongnate T., Sucharitakul J., Chaiyen P.

  Pyranose 2-oxidase (P2O) catalyzes the oxidation of aldopyranoses to form 2-keto sugars and H(2)O(2) . In this study, the mechanistic role of the conserved residues His548 and Asn593 in P2O was investigated by using site-directed mutagenesis, transient kinetics, and pH-dependence studies. As singl ... >> More

  Pyranose 2-oxidase (P2O) catalyzes the oxidation of aldopyranoses to form 2-keto sugars and H(2)O(2) . In this study, the mechanistic role of the conserved residues His548 and Asn593 in P2O was investigated by using site-directed mutagenesis, transient kinetics, and pH-dependence studies. As single mutants of H548 resulted in mixed populations of noncovalently bound and covalently linked FAD, double mutants containing H167A were constructed, in which the covalent histidyl-FAD linkage was removed in addition to having the H548 mutation. Single mutants H548A, H548N, H548S, H548D and double mutants (with H167A) could not be reduced by D-glucose. For the H167A/H548R mutant, the flavin could be reduced by D-glucose with the reduction rate constant about 220 times lower than that of the H167A mutant. The pH-dependence studies of H167A/H548R indicated that the rate constant of flavin reduction increased about 360-fold upon a pH rise corresponding to pK(a) >10.1, whereas the reactions of the wild-type and H167A mutant enzymes were pH independent. Therefore, the data suggest that a pK(a) value of >10.1 in the mutant enzyme is associated with the Arg548 residue, and that this residue must be unprotonated to efficiently catalyze flavin reduction. The data imply that for the wild-type P2O, the conserved His548 should be unprotonated in the pH range studied. The unprotonated His548 can act as a general base to abstract the 2-hydroxyl proton of D-glucose and initiate hydride transfer from the substrate to the flavin. Studies of the single mutant N593H showed that the flavin reduction rate constant was 114 times lower than that of the wild-type enzyme and was pH independent, while the K(d) for D-glucose binding was 19 times greater. << Less

  Chembiochem 12:2577-2586(2011) [PubMed] [EuropePMC]