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Molecular dynamics simulations give insight into D-glucose dioxidation at C2 and C3 by Agaricus meleagris pyranose dehydrogenase.

ABSTRACT: The flavin-dependent sugar oxidoreductase pyranose dehydrogenase (PDH) from the plant litter-degrading fungus Agaricus meleagris oxidizes D-glucose (GLC) efficiently at positions C2 and C3. The closely related pyranose 2-oxidase (P2O) from Trametes multicolor oxidizes GLC only at position C2. Consequently, the electron output per molecule GLC is twofold for PDH compared to P2O making it a promising catalyst for bioelectrochemistry or for introducing novel carbonyl functionalities into sugars. The aim of this study was to rationalize the mechanism of GLC dioxidation employing molecular dynamics simulations of GLC-PDH interactions. Shape complementarity through nonpolar van der Waals interactions was identified as the main driving force for GLC binding. Together with a very diverse hydrogen-bonding pattern, this has the potential to explain the experimentally observed promiscuity of PDH towards different sugars. Based on geometrical analysis, we propose a similar reaction mechanism as in P2O involving a general base proton abstraction, stabilization of the transition state, an alkoxide intermediate, through interaction with a protonated catalytic histidine followed by a hydride transfer to the flavin N5 atom. Our data suggest that the presence of the two potential catalytic bases His-512 and His-556 increases the versatility of the enzyme, by employing the most suitably oriented base depending on the substrate and its orientation in the active site. Our findings corroborate and rationalize the experimentally observed dioxidation of GLC by PDH and its promiscuity towards different sugars.

SUBMITTER: Graf MM

PROVIDER: S-EPMC3657087 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Pyranose dehydrogenases (PDHs; EC 1.1.99.29; AA3_2) demonstrate ability to oxidize diverse carbohydrates. Previous studies of these enzymes have also uncovered substrate-dependent regioselectivity, along with potential to introduce more than one carbonyl into carbohydrate substrates. Enzymatic oxidation of carbohydrates facilitates their further derivatization or polymerization into bio-based chemicals and materials with higher value; accordingly, PDHs that show activity on xylooligosaccharides could offer a viable approach to extract higher value from hemicelluloses that are typically fragmented during biomass processing. In this study, AbPDH1 from Agaricus bisporus and AmPDH1 from Leucoagaricus meleagris were tested using linear xylooligosaccharides, along with xylooligosaccharides substituted with either arabinofuranosyl or 4-O-(methyl)glucopyranosyluronic acid residues with degree of polymerization of two to five. Reaction products were characterized by HPAEC-PAD to follow substrate depletion, UPLC-MS-ELSD to quantify the multiple oxidation products, and ESI-MSn to reveal oxidized positions. A versatile method based on product reduction using sodium borodeuteride, and applicable to carbohydrate oxidoreductases in general, was established to facilitate the identification and quantification of oxidized products. AbPDH1 activity toward the tested xylooligosaccharides was generally higher than that measured for AmPDH1. In both cases, activity values decreased with increasing length of the xylooligosaccharide and when using acidic rather than neutral substrates; however, AbPDH1 fully oxidized all linear xylooligosaccharides, and 60-100% of all substituted xylooligosaccharides, after 24 h under the tested reaction conditions. Oxidation of linear xylooligosaccharides mostly led to double oxidized products, whereas single oxidized products dominated in reactions containing substituted xylooligosaccharides. Notably, oxidation of specific secondary hydroxyls vs. the reducing end C-1 depended on both the enzyme and the substrate. For all substrates, however, oxidation by both AbPDH1 and AmPDH1 was clearly restricted to the reducing and non-reducing xylopyranosyl residues, where increasing the length of the xylooligosaccharide did not lead to detectable oxidation of internal xylopyranosyl substituents. This detailed analysis of AbPDH1 and AmPDH1 action on diverse xylooligosaccharides reveals an opportunity to synthesize bifunctional molecules directly from hemicellulose fragments, and to enrich for specific products through appropriate PDH selection.

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Molecular dynamics simulations give insight into D-glucose dioxidation at C2 and C3 by Agaricus meleagris pyranose dehydrogenase.

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