The Effect of CBM1 and Linker on the Oxidase, Peroxidase and Monooxygenase Activities of AA9 LPMOs: Insight into Their Correlation with the Nature of Reductants and Crystallinity of Celluloses.

This study explores the effect of carbohydrate-binding module 1 (CBM1) and the linker on the function of auxiliary activity 9 (AA9) lytic polysaccharide monooxygenases (LPMOs), with a particular focus on monooxygenase activity, using different crystallinity celluloses and electron donors. The tested C1/C4-oxidizing AA9 LPMOs exhibited higher oxidase and peroxidase activities compared to those of the C4-oxidizing AA9 LPMOs. While the presence of CBM1 promoted cellulose-binding affinity, it reduced the oxidase activity of modular AA9 LPMOs. The effect of CBM1 on peroxidase activity was variable and enzyme-specific. Its influence on monooxygenase activity was linked to the type of reductants and the crystallinity of celluloses. Overall, CBM1 improved the monooxygenase activity on high-, medium-, and low-crystallinity celluloses when ascorbic acid (AscA) was used as the electron donor. CBM1 also facilitated monooxygenase activity on high-crystallinity cellulose, but significantly inhibited monooxygenase activity on low-crystallinity cellulose when cellobiose dehydrogenase (CDH) was the electron donor. Linker truncation of LOMO9C enhanced the cellulose-binding affinity but decreased both the oxidase and peroxidase activities. Linker truncation also impacted the monooxygenase activity in both the AscA-AA9 LPMO and CDH-AA9 LPMO systems, though its effect was less pronounced compared to that of CBM1. This work provides new insights into the role of the reductant type and cellulose crystallinity in the functionality of CBM1 and the linker in AA9 LPMOs.