Abstract
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.
Highlights
Pyranose dehydrogenase (PDH; pdh1 gene; pyranose:acceptor oxidoreductase; EC 1.1.99.29; sequence UniProt: Q3L245_9AGAR [1]) from the litter-decomposing fungus Agaricus meleagris is an extracellular, monomeric flavin-dependent oxidoreductase, with one flavin adenine dinucleotide (FAD) prosthetic group covalently bound per polypeptide chain [2].Like several other fungal sugar oxidoreductases, AmPDH belongs to the glucose-methanol-choline (GMC) oxidoreductase family [1]; and as the fungal pyranose 2-oxidase from Trametes multicolor (TmP2O), AmPDH produces aldoketose or diketose derivatives from non-phosphorylated sugars [2]
PDHs can be differentiated from P2Os based on: i) PDH is a glycosylated, extracellularly secreted enzyme, whereas P2O is non-glycosylated and located in the hyphal periplasmic space; ii) PDH is rather inert towards oxygen, while P2O is a typical flavoprotein oxidase; and iii) PDH oxidizes D-glucose at both the C2 and C3 position, whereas P2O is strictly regioselective for the glucosyl C2 position [3,4,5,6]
Activity on 2- and 3-Fluorinated Glucose When mixing 3-fluoro-3-deoxy-D-glucose (3FG) and AmPDH, the reaction mixture monitored at 30 s after mixing was colorless (Fig. 2) showing that AmPDH was rapidly reduced, presumably by a hydride moiety at glucose C2
Summary
Pyranose dehydrogenase (PDH; pdh gene; pyranose:acceptor oxidoreductase; EC 1.1.99.29; sequence UniProt: Q3L245_9AGAR [1]) from the litter-decomposing fungus Agaricus meleagris (synonym Leucoagaricus meleagris, Agaricus praeclaresquamosus, California fungus) is an extracellular, monomeric flavin-dependent oxidoreductase, with one flavin adenine dinucleotide (FAD) prosthetic group covalently bound per polypeptide chain [2].Like several other fungal sugar oxidoreductases, AmPDH belongs to the glucose-methanol-choline (GMC) oxidoreductase family [1]; and as the fungal pyranose 2-oxidase from Trametes multicolor (TmP2O), AmPDH produces aldoketose or diketose derivatives from non-phosphorylated sugars [2]. PDHs can be differentiated from P2Os based on: i) PDH is a glycosylated, extracellularly secreted enzyme, whereas P2O is non-glycosylated and located in the hyphal periplasmic space; ii) PDH is rather inert towards oxygen, while P2O is a typical flavoprotein oxidase; and iii) PDH oxidizes D-glucose at both the C2 and C3 position, whereas P2O is strictly regioselective for the glucosyl C2 position [3,4,5,6]. Volc and co-workers showed that P2O is expressed mainly by wood-decaying white-rot fungi (e.g., Phanerochaete, Trametes etc.), while PDH expression is limited to the Agaricales [7], which are typical litter-degrading fungi that live in forest and grassland soil and are the primary decomposers of residual plant material (leaves, needles, twigs, bark, grass) in the uppermost soil layer. It should be noted that P2O-encoding genes were found and experimentally confirmed in some members of the genus Aspergillus, which does not degrade lignocellulose, notably in species where a glucose 1oxidase (GOX) is absent [8]
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