Work

Investigation Into the Role of Reductants and Cosubstrates in Lytic Polysaccharide Monooxygenase Thermothielavioides terrestris AA9E Binding to Cellulose by Single‐Molecule Imaging

Year: 2025

Type: article

Abstract: ABSTRACT Cellulose‐active Lytic Polysaccharide Monooxygenases (LPMO) facilitate plant cell wall deconstruction by attacking ordered regions of cellulose. In vitro, reductants (e.g., ascorbic acid) reduce LPMOs to Cu(I)‐LPMO, and hydrogen peroxide (H 2 O 2 ) serves as co‐substrate for oxidative cleavage of cellulose glycosidic bonds. Super‐resolution single‐molecule imaging by total internal reflection fluorescence microscopy was used to visualize and enumerate binding events of fluorescently‐labeled Thermothielavioides terrestris AA9E ( Tt AA9E) on highly ordered cellulose fibrils in oxygen‐scavenging buffer systems. In the glucose oxidase/catalase (GODCAT) system, oxygen is converted to H 2 O 2 , then removed by catalase. Adding ascorbic acid to the GODCAT system promoted rapid binding to cellulose by Tt AA9E. In contrast, absent both oxygen and H 2 O 2 in the protocatechuic acid/protocatechuate 3,4‐dioxygenase (PCA/PCD) oxygen‐scavenging system, adding ascorbic acid nearly eliminated cellulose binding by Tt AA9E. Our results suggest that in the GODCAT system, Tt AA9Es are reduced by ascorbic acid and activated by H 2 O 2 , facilitating binding to cellulose. In the PCA/PCD system, reduced Tt AA9Es are not activated due to the lack of H 2 O 2 , suggesting that reduced Cu(I)‐ Tt AA9E cannot bind to cellulose without H 2 O 2 . Notably, in the PCA/PCD system with ascorbic acid, oxidized sugar release initially lagged but was observed at longer reaction times, suggesting that H 2 O 2 could be a limiting reactant generated in situ as oxygen becomes absorbed into solution. Binding durations of LPMO to cellulose were independent of experimental conditions: ( 82% ± 6%) of cellulose‐bound LPMOs resided briefly for 14 ± 2.5 s, while 16% ± 5% of the bound enzymes remained for 60 ± 9 s.

Source: Biotechnology and Bioengineering

DOI: 10.1002/bit.70080

Authors: Benedikt M. Blossom, Peter M. Goodwin, Camilla Fløien Angeltveit, Svein Jarle Horn, Alex O. Hitomi, Tina Jeoh