pH-Dependent Relationship between Catalytic Activity and Hydrogen Peroxide Production Shown via Characterization of a Lytic Polysaccharide Monooxygenase from Gloeophyllum trabeum

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that perform oxidative cleavage of recalcitrant polysaccharides. We have purified and characterized a recombinant family AA9 LPMO, LPMO9B, from ( LPMO9B) which is active on both cellulose and xyloglucan. Activity of the enzyme was tested in the presence of three different reductants: ascorbic acid, gallic acid, and 2,3-dihydroxybenzoic acid (2,3-DHBA). Under standard aerobic conditions typically used in LPMO experiments, the first two reductants could drive LPMO catalysis whereas 2,3-DHBA could not. In agreement with the recent discovery that H O can drive LPMO catalysis, we show that gradual addition of H O allowed LPMO activity at very low, substoichiometric (relative to products formed) reductant concentrations. Most importantly, we found that while 2,3-DHBA is not capable of driving the LPMO reaction under standard aerobic conditions, it can do so in the presence of externally added H O At alkaline pH, 2,3-DHBA is able to drive the LPMO reaction without externally added H O , and this ability overlaps entirely the endogenous generation of H O by LPMO9B-catalyzed oxidation of 2,3-DHBA. These findings support the notion that H O is a cosubstrate of LPMOs and provide insight into how LPMO reactions depend on, and may be controlled by, the choice of pH and reductant. Lytic polysaccharide monooxygenases promote enzymatic depolymerization of lignocellulosic materials by microorganisms due to their ability to oxidatively cleave recalcitrant polysaccharides. The properties of these copper-dependent enzymes are currently of high scientific and industrial interest. We describe a previously uncharacterized fungal LPMO and show how reductants, which are needed to prime the LPMO by reducing Cu(II) to Cu(I) and to supply electrons during catalysis, affect enzyme efficiency and stability. The results support claims that H O is a natural cosubstrate for LPMOs by demonstrating that when certain reductants are used, catalysis can be driven only by H O and not by O Furthermore, we show how auto-inactivation resulting from endogenous generation of H O in the LPMO-reductant system may be prevented. Finally, we identified a reductant that leads to enzyme activation without any endogenous H O generation, allowing for improved control of LPMO reactivity and providing a valuable tool for future LPMO research.