Agaricus meleagris pyranose dehydrogenase: Influence of covalent FAD linkage on catalysis and stability

Krondorfer, Iris; Brugger, Dagmar; Paukner, Regina; Scheiblbrandner, Stefan; Pirker, Katharina F.; Hofbauer, Stefan; Furtmüller, Paul G.; Obinger, Christian; Haltrich, Dietmar; Peterbauer, Clemens K.

doi:10.1016/j.abb.2014.07.008

Cited by 9 publications

(15 citation statements)

References 30 publications

(42 reference statements)

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“…The observed intermediate for variant H556A had an absorption maximum at 368 nm (Fig. C, dashed line) and was detected previously in the resting state of Am PDH, which could be attributed to an equilibrium of the oxidized form of the enzyme with its reduced forms . For variant H556A,

E^{normalo'}

was +84 ± 4 mV at pH 7.0 and 25 °C (Table ).…”

Section: Resultssupporting

confidence: 79%

“…whether the rate constants are significantly different for C2 or C3 oxidation). As reported previously, AmPDH is reduced rapidly by GLC at 30°C, with an apparent bimolecular rate constant (k app,30°C ) of 1.1 AE 0.03 9 10 5 M À1 Ás À1 [20]. Consequently, all stopped-flow experiments in the present study were conducted at 4°C to slow down the reaction and capture as much kinetic information as possible.…”

Section: C2 and C3 Oxidation Of Various Sugar Substrates Proceed Withmentioning

confidence: 55%

“…As described above, a k app,4°C of 5.4 AE 0.11 9 10 4 M À1 Ás À1 was obtained for the reductive half-reaction of AmPDH with GLC (Table 6), corresponding to a two-fold decrease compared to measurements at 30°C [20]. For the oxidative half-reaction of AmPDH with BQ, the first phase k obs(1) of the double-exponential fit corresponds to FAD oxidation with a large absorbance increase at 463 nm ( Fig.…”

Section: Variantmentioning

confidence: 61%

“…A T m value of 73.5°C was determined for recombinant wildtype AmPDH. Interestingly, variant H103A has a T m of 69.5°C, which is 5.7°C higher compared to the H103Y variant studied previously [20]. Apparently, the introduction of the bulkier tyrosine side chain at this position destabilizes the protein more significantly.…”

Section: Molecular Propertiesmentioning

confidence: 71%

“…To identify the rate‐limiting step in variant H556A and compare the results with recombinant wild‐type Am PDH, the transient‐state kinetics of their reductive (FAD → FADH 2 ) and oxidative (FADH 2 → FAD) half‐reactions were investigated by stopped‐flow spectroscopy, using GLC as electron donor and 1,4‐benzoquinone (BQ) as electron acceptor, respectively. In a previous study, the ferrocenium ion (Fc + ) was used as electron acceptor, which has an absorbance at 330–350 nm and 625 nm . Therefore, Fc + interferes to a greater extent with the flavin absorbance than BQ, which has an absorption maximum at 244 nm, and the corresponding hydroquinone at 222 nm and 296 nm, respectively .…”

Section: Resultsmentioning

confidence: 99%

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Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates

et al. 2015

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Monomeric Agaricus meleagris pyranose dehydrogenase (Am PDH) belongs to the glucose–methanol–choline family of oxidoreductases. An FAD cofactor is covalently tethered to His103 of the enzyme. Am PDH can double oxidize various mono‐ and oligosaccharides at different positions (C1 to C4). To study the structure/function relationship of selected active‐site residues of Am PDH pertaining to substrate (carbohydrate) turnover in more detail, several active‐site variants were generated, heterologously expressed in Pichia pastoris, and characterized by biochemical, biophysical and computational means. The crystal structure of Am PDH shows two active‐site histidines, both of which could take on the role as the catalytic base in the reductive half‐reaction. Steady‐state kinetics revealed that His512 is the only catalytic base because H512A showed a reduction in (k cat/KM)glucose by a factor of 105, whereas this catalytic efficiency was reduced by two or three orders of magnitude for His556 variants (H556A, H556N). This was further corroborated by transient‐state kinetics, where a comparable decrease in the reductive rate constant was observed for H556A, whereas the rate constant for the oxidative half‐reaction (using benzoquinone as substrate) was increased for H556A compared to recombinant wild‐type Am PDH. Steady‐state kinetics furthermore indicated that Gln392, Tyr510, Val511 and His556 are important for the catalytic efficiency of PDH. Molecular dynamics (MD) simulations and free energy calculations were used to predict d‐glucose oxidation sites, which were validated by GC‐MS measurements. These simulations also suggest that van der Waals interactions are the main driving force for substrate recognition and binding.

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E^{normalo'}

was +84 ± 4 mV at pH 7.0 and 25 °C (Table ).…”

Section: Resultssupporting

confidence: 79%

Section: C2 and C3 Oxidation Of Various Sugar Substrates Proceed Withmentioning

confidence: 55%

Section: Variantmentioning

confidence: 61%

Section: Molecular Propertiesmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

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Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates

et al. 2015

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Amino Acid Residues Controlling Domain Interaction and Interdomain Electron Transfer in Cellobiose Dehydrogenase

Motycka,

Csarman,

Rupp

et al. 2023

ChemBioChem

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The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD‐containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure‐based engineering. The electron transfer kinetics of wild‐type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped‐flow spectrophotometry and structural effects were studied by small‐angle X‐ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway – its mutation reducing the interdomain electron transfer 10‐fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

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

Graf

Weber

Kracher

et al. 2016

Appl Microbiol Biotechnol

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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.

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Agaricus meleagris pyranose dehydrogenase: Influence of covalent FAD linkage on catalysis and stability

Cited by 9 publications

References 30 publications

Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates

Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates

Amino Acid Residues Controlling Domain Interaction and Interdomain Electron Transfer in Cellobiose Dehydrogenase

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

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