Comparative Study of Two Chitin-Active and Two Cellulose-Active AA10-Type Lytic Polysaccharide Monooxygenases

Abstract

Lytic polysaccharide monooxygenases (LPMOs), found in family 9 (previously GH61), family 10 (previously CBM33), and the newly discovered family 11 of auxiliary activities (AA) in the carbohydrate-active enzyme classification system, are copper-dependent enzymes that oxidize sp(3)-carbons in recalcitrant polysaccharides such as chitin and cellulose in the presence of an external electron donor. In this study, we describe the activity of two AA10-type LPMOs whose activities have not been described before and we compare in total four different AA10-type LPMOs with the aim of finding possible correlations between their substrate specificities, sequences, and EPR signals. EPR spectra indicate that the electronic environment of the copper varies within the AA10 family even though amino acids directly interacting with the copper atom are identical in all four enzymes. This variation seems to be correlated to substrate specificity and is likely caused by sequence variation in areas that affect substrate binding geometry and/or by variation in a cluster of conserved aromatic residues likely involved in electron transfer. Interestingly, EPR signals for cellulose-active AA10 enzymes were similar to those previously observed for cellulose-active AA9 enzymes. Mutation of the conserved phenylalanine positioned in close proximity to the copper center in AA10-type LPMOs to Tyr (the corresponding residue in most AA9-type LPMOs) or Ala, led to complete or partial inactivation, respectively, while in both cases the ability to bind copper was maintained. Moreover, substrate binding affinity and degradation ability seemed hardly correlated, further emphasizing the crucial role of the active site configuration in determining LPMO functionality.

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

“…The spin Hamiltonian values g z ϭ 2.267 and ͉A z ͉ ϭ 152 ϫ 10 Ϫ4 cm Ϫ1 obtained for the Cu 2ϩ -loaded full-length enzyme are very similar to those obtained previously for cellulose-oxidizing LPMOs in the AA9 and AA10 families analyzed at pH values varying from 5.0 to 6.5 (5,9,17,46) and indicate a type 2 copper active site. Spin quantification of Cu 2ϩ in Cu 2ϩ -loaded NcLPMO9C was consistent with one copper-binding site/monomer.…”

A family of starch-active polysaccharide monooxygenases

“…PMOs are thought to bind to the substrate with their flat active site surface, which orients the copper center for selective oxidation at the C1 or C4 position (6,16,22). Some bacterial chitin-binding proteins are cellulose-active PMOs (8,13,14), further suggesting that the set of PMO substrates is restricted to β(1→4) linked polymers of glucose and glucose derivatives.Here, we report on the identification of new families of PMOs that contain several key features of previously characterized PMOs, but act on substrates different from cellulose or chitin. A member of one of these novel families of PMOs, NCU08746, was shown to oxidatively cleave amylose, amylopectin, and starch.…”

“…They have recently been found to oxidatively degrade chitin (6-8) and cellulose (8)(9)(10)(11)(12)(13)(14). PMOs have been shown to oxidize either the C1 or C4 atom of the β(1→4) glycosidic bond on the surface of chitin (6, 7) or cellulose (10)(11)(12)14), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases.…”

“…There are three families of PMOs characterized thus far: fungal PMOs that oxidize cellulose (9-12) (also known as GH61 and AA9); bacterial PMOs that are active either on chitin (6,8) or cellulose (8,13) (also known as CBM33 and AA10); and fungal PMOs that oxidize chitin (AA11) (7). Sequence homology between these three families is very low.…”

“…PMOs are thought to bind to the substrate with their flat active site surface, which orients the copper center for selective oxidation at the C1 or C4 position (6,16,22). Some bacterial chitin-binding proteins are cellulose-active PMOs (8,13,14), further suggesting that the set of PMO substrates is restricted to β(1→4) linked polymers of glucose and glucose derivatives.…”