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

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

Archives of biochemistry and biophysics 2014 Sep 15

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Pyranose dehydrogenase (PDH) is a monomeric flavoprotein belonging to the glucose-methanol-choline (GMC) family of oxidoreductases. It catalyzes the oxidation of free, non-phosphorylated sugars to the corresponding keto sugars. The enzyme harbors an FAD cofactor that is covalently attached to histidine 103 via an 8α-N(3) histidyl linkage. Our previous work showed that variant H103Y was still able to bind FAD (non-covalently) and perform catalysis but steady-state kinetic parameters for several substrates were negatively affected. In order to investigate the impact of the covalent FAD attachment in Agaricus meleagris PDH in more detail, pre-steady-state kinetics, reduction potential and stability of the variant H103Y in comparison to the wild-type enzyme were probed. Stopped-flow analysis revealed that the mutation slowed down the reductive half-reaction by around three orders of magnitude whereas the oxidative half-reaction was affected only to a minor degree. This was reflected by a decrease in the standard reduction potential of variant H103Y compared to the wild-type protein. The existence of an anionic semiquinone radical in the resting state of both the wild-type and variant H103Y was demonstrated using electron paramagnetic resonance (EPR) spectroscopy and suggested a higher mobility of the cofactor in the variant H103Y. Unfolding studies showed significant negative effects of the disruption of the covalent bond on thermal and conformational stability. The results are discussed with respect to the role of covalently bound FAD in catalysis and stability. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Citation

Iris Krondorfer, Dagmar Brugger, Regina Paukner, Stefan Scheiblbrandner, Katharina F Pirker, Stefan Hofbauer, Paul G Furtmüller, Christian Obinger, Dietmar Haltrich, Clemens K Peterbauer. Agaricus meleagris pyranose dehydrogenase: influence of covalent FAD linkage on catalysis and stability. Archives of biochemistry and biophysics. 2014 Sep 15;558:111-9