Volume 25, Issue 17 p. 4460-4471
Full Paper

The Mechanism of Sugar C−H Bond Oxidation by a Flavoprotein Oxidase Occurs by a Hydride Transfer Before Proton Abstraction

Thanyaporn Wongnate

Corresponding Author

Thanyaporn Wongnate

School of Biomolecular Science & Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Wangchan Valley, Rayong, 21210 Thailand

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Panida Surawatanawong

Panida Surawatanawong

Department of Chemistry and Center of Excellence, for Innovation in Chemistry, Mahidol University, Bangkok, 10400 Thailand

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Litavadee Chuaboon

Litavadee Chuaboon

Department of Biochemistry and Center for Excellence, in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand

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Narin Lawan

Narin Lawan

Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, 50200 Thailand

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Pimchai Chaiyen

Pimchai Chaiyen

School of Biomolecular Science & Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Wangchan Valley, Rayong, 21210 Thailand

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First published: 28 January 2019
Citations: 9

Graphical Abstract

Which is the first step on the path? Combined approaches using density functional theory (DFT) analysis and transient kinetics were employed to investigate the reaction mechanism of C−H bond oxidation in d-glucose, catalyzed by the enzyme pyranose 2-oxidase (P2O) (see figure).

Abstract

Understanding the reaction mechanism underlying the functionalization of C−H bonds by an enzymatic process is one of the most challenging issues in catalysis. Here, combined approaches using density functional theory (DFT) analysis and transient kinetics were employed to investigate the reaction mechanism of C−H bond oxidation in d-glucose, catalyzed by the enzyme pyranose 2-oxidase (P2O). Unlike the mechanisms that have been conventionally proposed, our findings show that the first step of the C−H bond oxidation reaction is a hydride transfer from the C2 position of d-glucose to N5 of the flavin to generate a protonated ketone sugar intermediate. The proton is then transferred from the protonated ketone intermediate to a conserved residue, His548. The results show for the first time how specific interactions around the sugar binding site promote the hydride transfer and formation of the protonated ketone intermediate. The DFT results are also consistent with experimental results including the enthalpy of activation obtained from Eyring plots, as well as the results of kinetic isotope effect and site-directed mutagenesis studies. The mechanistic model obtained from this work may also be relevant to other reactions of various flavoenzyme oxidases that are generally used as biocatalysts in biotechnology applications.

Conflict of interest

The authors declare no conflict of interest.