Regioselective C4 and C6 Double Oxidation of Cellulose by Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic degradation of hard-to-convert polysaccharides, such as chitin and cellulose. It is widely accepted that LPMOs catalyze a single regioselective oxidation of the C1 or C4 carbon of a glycosidic linkage, after which the destabilized linkage breaks. We discovered a series of novel C4/C6 double oxidized cello-oligosaccharides. Products were characterized, aided by sodium borodeuteride reduction, and hydrophilic interaction chromatography coupled to mass spectrometric analysis. The C4/C6 double oxidized products were generated by C4 and C1/C4 oxidizing LPMOs, but not by C1 oxidizing ones. By performing incubation and reduction in H 2 18 O, we confirmed that the C6 gem-diol structure resulted from oxygenation, though oxidation to a C6 aldehyde, followed by hydration to the C6 gem-diol, could not be excluded. Our findings can be extended to how the reactive LPMO-cosubstrate complex is positioned towards the substrate.