Enzymatic debranching is a key determinant of the xylan-degrading activity of family AA9 lytic polysaccharide monooxygenases

Abstract

Background Previous studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions. Results Using mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan. Furthermore, experiments with mixtures of PASC and arabinoglucuronoxylan from spruce showed that debranching of xylan with the GH62 arabinofuranosidase and a GH115 glucuronidase promoted LPMO activity. Analyses of mixtures with PASC and (non-arabinosylated) beechwood glucuronoxylan showed that GH115 action promoted LPMO activity also on this xylan. Remarkably, when WAX was incubated with Avicel instead of PASC in the presence of the GH62, both xylan and cellulose degradation by the LPMO9 were impaired, showing that the formation of cellulose–xylan complexes and their susceptibility to LPMO action also depend on the properties of the cellulose. These debranching effects not only relate to modulation of the cellulose–xylan interaction, which influences the conformation and rigidity of the xylan, but likely also affect the LPMO–xylan interaction, because debranching changes the architecture of the xylan surface. Conclusions Our results shed new light on xylanolytic LPMO9 activity and on the functional interplay and possible synergies between the members of complex lignocellulolytic enzyme cocktails. These findings will be relevant for the development of future lignocellulolytic cocktails and biomaterials.

Discovering Functional Diversity of Lytic Polysaccharide Monooxygenases from the Thermophilic Fungus Myceliophthora Thermophila and Their Application in Lignocellulosic Biomass Degradation

“…In particular, MtLPMO9G exhibited dual catalytic activities, degrading cellulose and xylan through oxidation and hydrolysis reactions, respectively. Most importantly, unlike reported AA9 LPMOs that catalyzed oxidative cleavage of glycosidic bonds in the xylan backbone in the presence of cellulose, 20,43,44 MtLPMO9G could able to independently hydrolyze xylan. Taken together, these discovered catalytic properties of LPMOs indicated that AA9 LPMOs from M. thermophila could attack cellulosic and hemicellulosic components of lignocellulosic biomass through multiple approaches, providing novel insights into how M. thermophila efficiently broke down the complex three-dimensional recalcitrant structure of lignocellulosic biomass.…”

“…11,36,37 Specifically, AA9 LPMOs exhibit activity towards cellulose and hemicellulose with β-1,4-linked glucose backbone. 20 AA14 LPMOs are active on xylan coated cellulose fibrils. 38 AA16 LPMOs boost AA9 LPMOs activity for cellulose degradation.…”

“…17−19 Until now AA9 LPMOs have been reported to be active on a wide range of polysaccharide substrates such as cellulose, xylan, xyloglucan, and glucomannan. 11,20,21 However, there is a lack of comprehensive and systematic studies on the functional diversity of AA9 LPMOs at the organism level. The biological role of abundant highly expressed AA9 LPMOs in single species for lignocellulosic biomass deconstruction remains to be elucidated.…”

“…LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of electron donors and co-substrates O 2 /H 2 O 2 , widely distributed in fungi, bacteria, viruses, plants, and insects. − According to the carbohydrate active enzyme classification system, LPMOs are classified into multiple auxiliary activity (AA) families, including AA9–AA11 and AA13–AA17. , AA9 LPMOs are the largest family of LPMOs, with more than 20,000 sequences encoding AA9 LPMOs found in the JGI database. , Significantly, dozens of copies of the AA9 LPMO gene exist in the genomes of most fungi, particularly ascomycetes and basidiomycetes. Not only that, many studies have shown that AA9 LPMOs are among the most highly expressed enzymes for filamentous fungi during lignocellulosic biomass deconstruction. − Until now AA9 LPMOs have been reported to be active on a wide range of polysaccharide substrates such as cellulose, xylan, xyloglucan, and glucomannan. ,, However, there is a lack of comprehensive and systematic studies on the functional diversity of AA9 LPMOs at the organism level. The biological role of abundant highly expressed AA9 LPMOs in single species for lignocellulosic biomass deconstruction remains to be elucidated.…”

Impact of the Copper Second Coordination Sphere on Catalytic Performance and Substrate Specificity of a Bacterial Lytic Polysaccharide Monooxygenase

“…The breakdown of polysaccharides is central to many biological processes and involves multiple enzymes, including lytic polysaccharide monooxygenases (LPMOs). These powerful monocopper enzymes are capable of oxidizing C–H bonds at the C1 and/or C4 carbon of glycosidic linkages in a broad range of polysaccharide substrates, including cellulose, − chitin, various types of hemicelluloses, , and starch. , Recently, these enzymes have also been shown to play a role in microbial pathogenesis , and cellular development. − …”