Thanyaporn Wongnate

Kinetic mechanism of pyranose 2-oxidase from trametes multicolor.

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.

The 1.6 Å crystal structure of pyranose dehydrogenase from Agaricus meleagris rationalizes substrate specificity and reveals a flavin intermediate.

Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.

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

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.

Kinetic isotope effects on the noncovalent flavin mutant protein of pyranose 2-oxidase reveal insights into the flavin reduction mechanism.

Pyranose 2-oxidase (P2O) from Trametes multicolor contains a flavin adenine dinucleotide (FAD) cofactor covalently linked to the N(3) atom of His167. The enzyme catalyzes the oxidation of aldopyranoses by molecular oxygen to generate 2-keto-aldoses and H(2)O(2) as products. In this study, the transient kinetics and primary and solvent kinetic isotope effects of the mutant in which His167 has been replaced with Ala (H167A) were investigated, to elucidate the functional role of the 8a-N(3)-histidyl FAD linkage and to gain insights into the reaction mechanism of P2O. The results indicate that the covalent linkage is mainly important for a reductive half-reaction in which the FAD cofactor is reduced by d-glucose, while it is not important for an oxidative half-reaction in which oxygen reacts with the reduced FAD to generate H(2)O(2). d-Glucose binds to H167A via multiple binding modes before the formation of the active Michaelis complex, and the rate constant of flavin reduction decreases approximately 22-fold compared to that of the wild-type enzyme. The reduction of H167A using d-glucose isotopes (2-d-d-glucose, 3-d-d-glucose, and 1,2,3,4,5,6,6-d(7)-d-glucose) as substrates indicates that the primary isotope effect results only from substitution at the C2 position, implying that H167A catalyzes the oxidation of d-glucose regiospecifically at this position. No solvent kinetic isotope effect was detected during the reductive half-reaction of the wild-type or H167A enzyme, implying that the deprotonation of the d-glucose C2-OH group may occur readily upon the binding to P2O and is not synchronized with the cleavage of the d-glucose C2-H bond. The mutation has no drastic effect on the oxidative half-reaction of P2O, as H167A is very similar to the wild-type enzyme with respect to the kinetic constants and the formation of the C4a-hydroperoxyflavin intermediate. Kinetic mechanisms for both half-reactions of H167A were proposed on the basis of transient kinetic data and were verified by kinetic simulations and steady-state kinetic parameters.

Hydrogen Peroxide Elimination from C4a-hydroperoxyflavin in a Flavoprotein Oxidase Occurs through a Single Proton Transfer from Flavin N5 to a Peroxide Leaving Group

C4a-hydroperoxyflavin is found commonly in the reactions of flavin-dependent monooxygenases, in which it plays a key role as an intermediate that incorporates an oxygen atom into substrates. Only recently has evidence for its involvement in the reactions of flavoprotein oxidases been reported. Previous studies of pyranose 2-oxidase (P2O), an enzyme catalyzing the oxidation of pyranoses using oxygen as an electron acceptor to generate oxidized sugars and hydrogen peroxide (H2O2), have shown that C4a-hydroperoxyflavin forms in P2O reactions before it eliminates H2O2 as a product (Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Biochemistry 47, 8485–8490). In this report, the solvent kinetic isotope effects (SKIE) on the reaction of reduced P2O with oxygen were investigated using transient kinetics. Our results showed that D2O has a negligible effect on the formation of C4a-hydroperoxyflavin. The ensuing step of H2O2 elimination from C4a-hydroperoxyflavin was shown to be modulated by an SKIE of 2.8 ± 0.2, and a proton inventory analysis of this step indicates a linear plot. These data suggest that a single-proton transfer process causes SKIE at the H2O2 elimination step. Double and single mixing stopped-flow experiments performed in H2O buffer revealed that reduced flavin specifically labeled with deuterium at the flavin N5 position generated kinetic isotope effects similar to those found with experiments performed with the enzyme pre-equilibrated in D2O buffer. This suggests that the proton at the flavin N5 position is responsible for the SKIE and is the proton-in-flight that is transferred during the transition state. The mechanism of H2O2 elimination from C4a-hydroperoxyflavin is consistent with a single proton transfer from the flavin N5 to the peroxide leaving group, possibly via the formation of an intramolecular hydrogen bridge.

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

Pyranose 2‐oxidase (P2O) catalyzes the oxidation of aldopyranoses to form 2‐keto sugars and H2O2. 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 pKa>10.1, whereas the reactions of the wild‐type and H167A mutant enzymes were pH independent. Therefore, the data suggest that a pKa 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 Kd for D‐glucose binding was 19 times greater.

H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase

Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme-flavin bond helps to increase the flavins redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme-flavin covalent bond--the flavinylation reaction--has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl-flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand.

Reduction kinetics of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M(-1) s(-1). In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s(-1). At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is ~51 s(-1), whereas without 3-HB, the rate constant is 0.43 s(-1) at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme-product (2,5-DHB) complex, with a rate constant of 45 ± 2 s(-1). The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because Em 0 for the enzyme-substrate complex is -179 ± 1 mV compared to an E(m)(0) of -175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme-ligand complex to a fully activated form before flavin reduction takes place.

Magnetic field effects as a result of the radical pair mechanism are unlikely in redox enzymes.

Environmental exposure to electromagnetic fields is potentially carcinogenic. The radical pair mechanism is considered the most feasible mechanism of interaction between weak magnetic fields encountered in our environment and biochemical systems. Radicals are abundant in biology, both as free radicals and reaction intermediates in enzyme mechanisms. The catalytic cycles of some flavin-dependent enzymes are either known or potentially involve radical pairs. Here, we have investigated the magnetic field sensitivity of a number of flavoenzymes with important cellular roles. We also investigated the magnetic field sensitivity of a model system involving stepwise reduction of a flavin analogue by a nicotinamide analogue—a reaction known to proceed via a radical pair. Under the experimental conditions used, magnetic field sensitivity was not observed in the reaction kinetics from stopped-flow measurements in any of the systems studied. Although widely implicated in radical pair chemistry, we conclude that thermally driven, flavoenzyme-catalysed reactions are unlikely to be influenced by exposure to external magnetic fields.

Enzymatic fuel cells with an oxygen resistant variant of pyranose-2-oxidase as anode biocatalyst

In enzymatic fuel cells (EnFCs), hydrogen peroxide formation is one of the main problems when enzymes, such as, glucose oxidase (GOx) is used due to the conversion of oxygen to hydrogen peroxide in the catalytic reaction. To address this problem, we here report the first demonstration of an EnFC using a variant of pyranose-2-oxidase (P2O-T169G) which has been shown to have low activity towards oxygen. A simple and biocompatible immobilisation approach incorporating multi-walled-carbon nanotubes within ferrocene (Fc)-Nafion film was implemented to construct EnFCs. Successful immobilisation of the enzymes was demonstrated showing 3.2 and 1.7-fold higher current than when P2O-T169G and GOx were used in solution, respectively. P2O-T169G showed 25% higher power output (maximum power density value of 8.45 ± 1.6 μW cm-2) and better stability than GOx in aerated glucose solutions. P2O-T169G maintained > 70% of its initial current whereas GOx lost activity > 90% during the first hour of 12 h operation at 0.15 V (vs Ag/Ag+). A different fuel cell configuration using gas-diffusion cathode and carbon paper electrodes were used to improve the power output of the fuel cell to 29.8 ± 6.1 µW cm-2. This study suggests that P2O-T169G with low oxygen activity could be a promising anode biocatalyst for EnFC applications.