A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase

Abstract

(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.

Stabilization of C4a-Hydroperoxyflavin in a Two-component Flavin-dependent Monooxygenase Is Achieved through Interactions at Flavin N5 and C4a Atoms

“…10) (41). For pyranose 2-oxidase, mutations of Thr-169, a residue that is located ϳ3 Å away from the flavin N5 similar to Ser-171 in C 2 , abolished the formation of C4a-hydroperoxyflavin (46). Based on this view, we predict that the role of Ser-171 in C 2 is for the hydrogen bonding interaction with flavin N5-H to impede the deprotonation at the flavin N5-H. At low pH (Fig.…”

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

“…In Thr 169 mutants, the optimum H-bonding interaction between the O ␥ of Thr 169 and the flavin N5 is removed, resulting in an environment with lower dielectric constants, which promotes the intra-molecular H-bridge formation and thus facilitates H 2 O 2 elimination. Therefore, the abolishment of C4a-hydroperoxyflavin that is observed in Thr 169 mutants may be due to an increased decay of the intermediate (31), that is, an increased rate constant of H 2 O 2 elimination. The model in Fig.…”

“…8 ] close enough to interact with the flavin N5) to Ser, Ala, or Gly, abolish the formation of the C4a-hydroperoxyflavin intermediate (Fig. 9A) (31). For the wild-type enzyme, the H-bond interaction between the flavin N5 and the O ␥ of the Thr 169 side chain may divert the interaction of the intramolecular H-bridge that facilitates H 2 O 2 elimination (Fig.…”

“…1) (12)(13)(14). The formation of C4a-hydroperoxy-FAD in P2O is known to require optimized interactions, especially around the FAD N5/C4a locus because mutations of Thr 169 to Ser/Ala/Asn/Gly resulted in the abolishment of the intermediate formation (31).…”

A survey of genes encoding H2O2-producing GMC oxidoreductases in 10 Polyporales genomes

“…P2O is produced by other Polyporales, including Trametes multicolor (5 Trametes ochracea) (Leitner et al 2001), and most recent P2O research focuses on this enzyme, whose reaction mechanisms have been elucidated in a variety of crystallographic, spectroscopic, directed mutagenesis, isotope labeling and kinetic studies (Hallberg et al 2004;Sucharitakul et al 2008;Prongjit et al 2009Prongjit et al , 2010Pitsawong et al 2010;Wongnate et al 2011Wongnate et al , 2014.…”