Kinetic Characterization of a Putatively Chitin-Active LPMO Reveals a Preference for Soluble Substrates and Absence of Monooxygenase Activity.

Vincent G.H Eijsink,Morten Sørlie,Lukas Rieder,Dejan Petrović,Priit Väljamäe

doi:10.1021/acscatal.1c03344

Abstract

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are recognized as important contributors to aerobic enzymatic degradation of recalcitrant polysaccharides such as chitin and cellulose. LPMOs are remarkably abundant in nature, with some fungal species possessing more than 50 LPMO genes, and the biological implications of this diversity remain enigmatic. For example, chitin-active LPMOs have been encountered in biological niches where chitin conversion does not seem to take place. We have carried out an in-depth kinetic characterization of a putatively chitin-active LPMO from Aspergillus fumigatus (AfAA11B), which, as we show here, has multiple unusual properties, such as a low redox potential and high oxidase activity. Furthermore, AfAA11B is hardly active on chitin, while being very active on soluble oligomers of N-acetylglucosamine. In the presence of chitotetraose, the enzyme can withstand considerable amounts of H2O2, which it uses to efficiently and stoichiometrically convert this substrate. The unique properties of AfAA11B allowed experiments showing that it is a strict peroxygenase and does not catalyze a monooxygenase reaction. This study shows that nature uses LPMOs for breaking glycosidic bonds in non-polymeric substrates in reactions that depend on H2O2. The quest for the true substrates of these enzymes, possibly carbohydrates in the cell wall of the fungus or its competitors, will be of major interest.

Highlights

Lytic polysaccharide monooxygenases (LPMOs) are receiving massive attention due to their ability to degrade recalcitrant polysaccharides, such as cellulose and chitin, in biomass conversion.[1−7] Through the use of powerful redox chemistry, lytic polysaccharide monooxygenases (LPMOs) are able to selectively activate C−H bonds that require overcoming an energy barrier of ∼100 kcal/mol.[3,8−11] LPMOs are abundant in nature and categorized, based on their sequences, in seven distinct families (AA9-AA11 and AA13-AA16), within the class of auxiliary activities (AAs) in the CAZy database.[12]
A. fumigatus expresses at least three AA11s where AfAA11B has low sequence identity with the other two, suggesting different biological roles
Previous work on AoAA11, with a similar domain structure and 72.6% sequence identity in the catalytic domain, suggested that this enzyme is involved in chitin degradation,[36] but functional characterization of AoAA11 was limited in this previous study

Summary

Introduction

Lytic polysaccharide monooxygenases (LPMOs) are receiving massive attention due to their ability to degrade recalcitrant polysaccharides, such as cellulose and chitin, in biomass conversion.[1−7] Through the use of powerful redox chemistry, LPMOs are able to selectively activate C−H bonds that require overcoming an energy barrier of ∼100 kcal/mol.[3,8−11] LPMOs are abundant in nature and categorized, based on their sequences, in seven distinct families (AA9-AA11 and AA13-AA16), within the class of auxiliary activities (AAs) in the CAZy database.[12]. Lytic polysaccharide monooxygenases (LPMOs) are receiving massive attention due to their ability to degrade recalcitrant polysaccharides, such as cellulose and chitin, in biomass conversion.[1−7] Through the use of powerful redox chemistry, LPMOs are able to selectively activate C−H bonds that require overcoming an energy barrier of ∼100 kcal/mol.[3,8−11] LPMOs are abundant in nature and categorized, based on their sequences, in seven distinct families In the oxygen-driven mechanism, a fundamental challenge is the thermodynamically unfavorable formation of superoxide through lowered reduction of by binding. Accumulating data from experiments and modeling indicate that the peroxygenase reaction entails homolytic cleavage of H2O2 by the reduced LPMO, leading to the formation of a hydroxyl radical that may react directly with the substrate or generate a Cu(II)-oxyl species.[11,15,25]

Methods

Results

Conclusion