Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris)

Abstract

Multigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucose–methanol–choline (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-8051-1) contains supplementary material, which is available to authorized users.

Multiplicity of enzymatic functions in the CAZy AA3 family

“…In contrast, their kinetic properties and relative activities differed significantly for model electron acceptor substrates studied, a radical (the ABTS cation radical), a quinone (benzoquinone), and a complexed iron ion (ferrocenium ion). Thus, a possible explanation for this multiplicity of PDH could be that in vivo the different PDH isoforms react preferentially with structurally different electron acceptors (Graf et al 2017 ).…”

“…Explanations for the existence of multigenic AA family members have been given as functional redundancy, functional diversification including subtle differences in substrate specificity of individual enzymes, or fine-tuning of the gene regulation/expression in order to adapt fungal organisms to the diversity of their natural substrates (Lenfant et al 2017 ; Levasseur et al 2013 ). Examples for this fine-tuning and differences in expression of AA 3 genes as a response to different growth conditions and substrates can be found in the literature, e.g., different expression patterns of AA3 members when growing the plant-pathogenic white-rot fungus Heterobasidion irregulare on heartwood and reaction-zone wood of Norway spruce (Yakovlev et al 2013 ), yet very few studies have looked at the biochemical properties and enzymological differences of individual AA3 family member isoforms from one single organism to better understand this multigenicity (Graf et al 2017 ; Mathieu et al 2016 ). Plant cell walls are very complex composite structures, and the multigenicity in the AA3 subfamilies might reflect necessities and adaptations to the degradation of these complex substrates.…”

Substrate Preference Pattern of Agaricus meleagris Pyranose Dehydrogenase Evaluated through Bioelectrochemical Flow Injection Amperometry

“…In the last decade Agaricus meleagris pyranose dehydrogenase ( Am PDH) has been extensively studied due to its potential role in synthesis of novel sugar and fine chemicals that are not available by chemical reaction. Pyranose dehydrogenase (PDH; EC 1.1.99.29) is an extracellular, monomeric glycosylated polypeptide, with one flavin adenine dinucleotide (FAD) prosthetic group covalently tethered to His103 of the enzyme . Unlike the homotetrameric structure of pyranose‐2 oxidase (POx), PDH has a single polypeptide structure with a high degree of glycosylation, varying between approximately 7 % in the natural A. meleagris enzyme to 30 % in the recombinantly expressed enzyme from Pichia pastoris .…”

Oxidoreductases and Reactive Oxygen Species in Conversion of Lignocellulosic Biomass

“…Red stars indicate the absence of kinetic data. Abbreviations (and associated references for corresponding data) are as follows: Lac, laccase(380)(381)(382)(383); POD, peroxidase(380,(384)(385)(386)(387)(388); CDH, cellobiose dehydrogenase(102,(128)(129)(130)(389)(390)(391)(392); GOX, glucose oxidase(167,(393)(394)(395)(396)(397)(398); AAO, aryl alcohol oxidase(166,399,400); GDH, glucose dehydrogenase(401,402); AAQO, aryl alcohol quinone oxidoreductase(403); PDH, pyranose dehydrogenase(404)(405)(406)(407); AOX, alcohol oxidase(177,(408)(409)(410); P2O, pyranose 2-oxidase(181,(411)(412)(413); VAO, vanillyl alcohol oxidase(186,187); GLOX, glyoxal oxidase(170,<...>…”