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

Abstract

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. Here, a series of novel C4/C6 double oxidized cello‐oligosaccharides was discovered. 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 H218O, it was confirmed that the C6 gem‐diol structure resulted from oxygenation, although oxidation to a C6 aldehyde, followed by hydration to the C6 gem‐diol, could not be excluded. These findings can be extended to how the reactive LPMO‐cosubstrate complex is positioned towards the substrate.

Comparison of Six Lytic Polysaccharide Monooxygenases from Thermothielavioides terrestris Shows That Functional Variation Underlies the Multiplicity of LPMO Genes in Filamentous Fungi

“…The introduction of a hydroxyl group at either the C 1 or C 4 position by the LPMO leads to spontaneous bond cleavage and formation of a lactone (C 1 oxidation) or a 4-ketoaldose (C 4 oxidation), which are in a pH-dependent equilibrium with their hydrated forms, being an aldonic acid or a gemidiol, respectively ( 9 , 18 ). In addition to cleavage of the glycosidic bond by C 1 and C 4 oxidation, C 6 oxidation of cellulose has also been reported ( 19 – 21 ) but needs further corroboration.…”

From Nature‐Sourced Polysaccharide Particles to Advanced Functional Materials

“…[ 91 ] Furthermore, non‐regioselective modification can also alter the enzymatic degradation profile of PS molecules, as most enzymatic catalyses require a specific moiety for recognition by the active sites of the enzymes, mediated by complementary shapes of the enzyme and the PS molecules. [ 92 ] The introduction of an arabinofuranosyl group at C3 position of xylan, for instance, protects the PS molecules from enzymatic degradation by β‐xylosidase. [ 93 ]…”

A Novel Approach to Analyze the Impact of Lytic Polysaccharide Monooxygenases (Lpmos)On Cellulosic Fibres

“…Note that some LPMOs, including TrAA9A and TaAA9A used in this study, are not strictly C1-oxidizing or C4oxidizing but produce mixtures of C1-and C4-oxidized products, which results in simultaneous generation of new non-reducing and reducing ends, respectively. Apart from that regular action mode, a certain amount of additional oxidized functional groups is expected to be introduced upon enzymatic treatment due to possible side activities of LPMOs (e.g., C6-oxidation (Bey et al, 2013;Chen et al, 2018;Sun et al, 2021)) and/or to non-specific oxidation, which may occur due to the presence of redox-active transition metal ions. In terms of newly introduced oxidized functionalities through the primary LPMO reaction, a C1-oxidizing LPMO would introduce one lactone (cyclic ester) functionality, whereas a C4-oxidizing LPMO would generate two carbonyls, the new reducing end (hemiacetal) and a ketone (hydrate) at C4.…”