Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H 2 O 2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose-and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k cat , and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Pyranose 2-oxidase (P2Ox, 3 pyranose:oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) is a flavin adenine dinucleotide (FAD)-dependent oxidase present in the hyphal periplasmic space (1) of wood-degrading basidiomycetes (2, 3). These fungi are the only known microorganisms that are capable of fully mineralizing lignin, and P2Ox has a proposed role in the oxidative events (4) of lignin degradation by providing the essential co-substrate, H 2 O 2 , for lignin and manganese peroxidases (5, 6). An alternative hypothesis assigns a role for P2Ox in both H 2 O 2 production and in the reduction of quinones in the periplasm or in the extracellular environment (7). P2Ox from the white-rot fungi Trametes multicolor (Trametes ochracea) and Peniophora gigantea are hitherto the most studied biochemically (7-10) and structurally (11, 12).P2Ox oxidizes a broad range of carbohydrate substrates that are natural constituents of hemicelluloses, allowing most lignocellulose-derived sugars to be utilized. Substrates can be oxidized regioselectively at the C2 position, although some oxidation at C3 can occur as a side reaction (10). For C2 oxidation, D-glucose, D-xylose, and L-sorbose are good or reasonably good substrates, and D-galactose and L-arabinose perform poorly as substrates (7). Based on the catalytic efficiency, k cat /K m , D-glucose (D-Glc) is the best substrate for T. multicolor P2Ox (7). Substrates that are oxidized at C3 were analyzed for P. gigantea P2Ox and include 2-deoxy-D-glucose, 2-keto-D-glucose, and methyl ␤-D-glucosides (13, 10). That oxidation can take place either at C2 or at C3 presupposes two distinct, productive binding modes (referred to here as C2 ox and C3 ox ) for a monosaccharide in the P2Ox active site.P2Ox from T. multicolor is homotetrameric with a molecular mass of 270 kDa (7) where each of the four subunits carries one FAD molecule bound covalently to N ⑀2 (i.e. N3) of His 167 via its 8␣-methyl group (14, 11). The...

Hydroxylation of aromatic compounds in bacteria by single component flavoprotein hydroxylases has been studied extensively for 40 years (1-3). Recently, several research groups, including ours, reported that hydroxylation of aromatic compounds can be catalyzed by two-protein or multiprotein monooxygenases. These include p-hydroxyphenylacetate 3-hydroxylase (HPAH) 3 from Pseudomonas putida (4), Escherichia coli (5), andAcinetobacter baumannii (6), phenol hydroxylase (PheA) from both Bacillus stearothermophilus BR219 and Bacillus thermoglucosidasius A7 (7, 8), chlorophenol-4-monooxygenase from Burkholderia cepacia AC1100 (9), 2,4,6-trichlorophenol monooxygenase from Ralstonia eutropha JMP134 (10), pyrrole-2-carboxylate monooxygenase from Rhodococcus sp (11), styrene monooxygenase from Pseudomonas VLB120 (12, 13), and p-nitrophenol hydroxylase from Bacillus sphaericus (14). Most of these enzyme systems consist of reductase and monooxygenase components, where the reductase component provides reduced flavin for the monooxygenase component to use for hydroxylating the aromatic substrate. The number of enzymes known in this class continues to increase and many more hypothetical proteins derived from genome projects have also been identified (15). Hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA) by HPAH is especially interesting because the same reaction is carried out by at least three types of two-component enzymes. The first HPAH purified was from P. putida, and it was shown to have FAD tightly bound to the smaller protein, and the larger protein (at that time) was thought to be a coupling protein enabling hydroxylation (4, 16). A different HPAH system was later isolated from E. coli W, and studies have shown that the smaller component (HpaC) is a flavin reductase that generates reduced FAD to be transferred to the larger component (HpaB) to hydroxylate HPA (5, 17). A detailed analysis of the mechanism of the E. coli-type HPAH is now in progress using the homologue from P. aeruginosa (18). The oxygenase in this system exhibits complex dynamics in catalysis (19).Our group has isolated HPAH from A. baumannii and shown that the enzyme is quite different from the analogous HPAH enzymes from either P. putida or E. coli (6,15,20). The A. baumannii HPAH is a two protein enzyme system consisting of a smaller reductase component (C 1 ) and a larger oxygenase component (C 2 ) (6). Sequence and several catalytic properties indicate that both components are different from others in the two protein class of aromatic hydroxylases (15,20). Our recent investigations of the reaction mechanisms of C 1 have shown that HPA controls the reduction of C 1 -bound FMN by NADH by shifting the enzyme into a more active conformation (20). By contrast, HPA has no effect at all on the activity of the reductase from the E. coli-type HPAH from P. aeruginosa (18). The HPAH from P. putida (above) (16) requires fresh examination based upon our current knowledge. It is possible that this enzyme system operates in ...

This work describes for the first time the identification of a reaction intermediate, C4a-hydroperoxyflavin, during the oxidative half-reaction of a flavoprotein oxidase, pyranose 2-oxidase (P2O) from Trametes multicolor, by using rapid kinetics. The reduced P2O reacted with oxygen with a forward rate constant of 5.8 x 10 (4) M (-1) s (-1) and a reverse rate constant of 2 s (-1), resulting in the formation of a C4a-hydroperoxyflavin intermediate which decayed with a rate constant of 18 s (-1). The absorption spectrum of the intermediate resembled the spectra of flavin-dependent monooxygenases. A hydrophobic cavity formed at the re side of the flavin ring in the closed state structure of P2O may help in stabilizing the intermediate.

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...

p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) from Acinetobacter baumannii catalyzes hydroxylation of HPA to form 3,4-dihydroxyphenylacetate. It is a two-protein system consisting of a smaller reductase component (C(1)) and a larger oxygenase component (C(2)). C(1) is a flavoprotein containing FMN, and its function is to provide reduced flavin for C(2) to hydroxylate HPA. We have shown here that HPA plays important roles in the reaction of C(1). The apoenzyme of C(1) binds to oxidized FMN tightly with a K(d) of 0.006 microM at 4 degrees C, but with a K(d) of 0.038 microM in the presence of HPA. Reduction of C(1) by NADH occurs in two phases with rate constants of 11.6 and 3.1 s(-)(1) and K(d) values for NADH binding of 2.1 and 1.5 mM, respectively. This result indicates that C(1) exists as a mixture of isoforms. However, in the presence of HPA, the reduction of C(1) by NADH occurred in a single phase at 300 s(-)(1) with a K(d) of 25 microM for NADH binding at 4 degrees C. Formation of the C(1)-HPA complex prior to binding of NADH was required for this stimulation. The redox potentials indicate that the rate enhancement is not due to thermodynamics (E degrees (m) of the C(1)-HPA complex is -245 mV compared to an E degrees (m) of C(1) of -236 mV). When the C(1)-HPA complex was reduced by 4(S)-NADH, the reduction rate was changed from 300 to 30 s(-)(1), giving a primary isotope effect of 10 and indicating that C(1) is specifically reduced by the pro-(S)-hydride. In the reaction of reduced C(1) with oxygen, the reoxidation reaction is also biphasic, consistent with reduced C(1) being a mixture of fast and slow reacting species. Rate constants for both phases were the same in the absence and presence of HPA, but in the presence of HPA, the equilibrium shifted toward the faster reacting species.

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). HPAH is composed of two proteins: a flavin mononucleotide (FMN) reductase (C1) and an oxygenase (C2). C1 catalyzes the reduction of FMN by NADH to generate reduced FMN (FMNH-) for use by C2 in the hydroxylation reaction. C1 is unique among the flavin reductases in that the substrate HPA stimulates the rates of both the reduction of FMN and release of FMNH- from the enzyme. This study quantitatively shows the kinetics of how the C1-bound FMN can be reduced and released to be used efficiently as the substrate for the C2 reaction; additional FMN is not necessary. Reactions in which O2 is rapidly mixed with solutions containing C1-FMNH- and C2 are very similar to those in which solutions containing O2 are mixed with one containing the C2-FMNH- complex. This suggests that in a mixture of the two proteins FMNH- binds more tightly to C2 and has already been completely transferred to C2 before it reacts with oxygen. Rate constants for the transfer of FMNH- from C1 to C2 were found to be 0.35 and >or=74 s-1 in the absence and presence of HPA, respectively. The reduction of cytochrome c by FMNH- was also used to measure the dissociation rate of FMNH- from C1. In the absence of HPA, FMNH- dissociates from C1 at 0.35 s-1, while with HPA present it dissociates at 80 s-1; these are the same rates as those for the transfer from C1 to C2. Therefore, the dissociation of FMNH- from C1 is rate-limiting in the intermolecular transfer of FMNH- from C1 to C2, and this process is regulated by the presence of HPA. This regulation avoids the production of H2O2 in the absence of HPA. Our findings indicate that no protein-protein interactions between C1 and C2 are necessary for efficient transfer of FMNH- between the proteins; transfer can occur by a rapid-diffusion process, with the rate-limiting step being the release of FMNH- from C1.

Edited by F. Peter GuengerichThe accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

(D-Glc) and several aldopyranoses at the C2 position to yield the corresponding 2-ketoaldoses and hydrogen peroxide (2). The overall catalytic reaction can be divided into two half-reactions (Scheme 1) obeying a Ping-Pong type mechanism at pH 7 (3): a reductive half-reaction in which the protein-bound flavin receives a hydride equivalent from a sugar substrate to produce the reduced FAD (FADH Ϫ ) and the 2-keto-sugar, and an oxidative half-reaction in which two electrons are transferred from the reduced flavin to O 2 to form H 2 O 2 (2, 3).We reported the first detection of a C4a-hydroperoxyflavin intermediate for a flavoprotein oxidase (4). This flavin intermediate has not been previously observed for flavoprotein oxidases but has been limited to reactions of flavoprotein monooxygenases (5-7). Besides P2O, an intermediate in flavoprotein oxidases has been detected only in the crystalline forms of choline oxidase (1.86 Å) (8) and nitroalkane oxidase (in the presence of cyanide) (9), and in the mutant form, C42S, of an NADH oxidase (10). In the case of glucose oxidase from Aspergillus niger, the generation of a flavin semiquinone-superoxide radical pair using pulse radiolysis resulted in formation of a putative C4a-hydroperoxyflavin intermediate (11). Studies on the P2O reductive half-reaction indicate that P2O binds D-Glc according to a two-step binding mechanism: first an initial complex is formed, followed by an isomerization step to form an active Michaelis ES complex (P2O⅐glucose). Interestingly, this complex shows higher absorbance at 395 nm than does the oxidized enzyme, which is unique among flavoprotein oxidases (3).P2O belongs to the large family of glucose-methanol-choline oxidoreductases (12, 13) with the catalytic side chains His 548 - * This work was supported in part by the Thailand Research Fund throughGrants BRG5180002 (to P. C.), MRG4980117 (to J. S.), and PHD/0151/2547 of the Royal Golden Jubilee Ph.D. program (to M. P.) and by the Faculty of Science, Mahidol University (to P. C.) and from the Faculty of Dentistry Chulalongkorn University (to J. S.). The atomic coordinates and structure factors (codes 3K4B and 3K4C) The abbreviations used are: P2O, pyranose 2-oxidase; ABTS, 2,2Ј-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; , wavelength; WT, wild type; Mes, 4-morpholineethanesulfonic acid.