Background: Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that cleave polysaccharides. Results: We describe a novel LPMO and use a range of analytical methods to characterize its activity. Conclusion: Cellulose and cello-oligosaccharides are cleaved by oxidizing the sugar at the nonreducing end in the C4 position. Significance: This study provides unequivocal evidence for C4 oxidation of the nonreducing end sugar and demonstrates a novel LPMO substrate specificity.

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.biorefinery | metallo enzymes | GH61 | CBM33

While it has been known for decades that enzymatic oxidation of lignin by laccases and peroxidases plays a role in microbial biomass conversion of lignin, it has only very recently become apparent that oxidative processes also play a major role in the conversion of polysaccharides. The latter process is carried out by so-called Lytic Polysaccharide MonoOxygenases (LPMOs) 1 , which are copper-dependent enzymes capable of breaking glycosidic bonds in polysaccharides, such as cellulose, xyloglucan, glucomannan, xylan, starch and chitin 2-9 . LPMO activity depends on the presence of molecular oxygen and requires an electron donor. So far, it has been shown that electrons can be provided by cellobiose dehydrogenase (CDH) 10 or by small molecule electron donors such as ascorbic acid 7 or gallic acid 6 . However, virtually nothing is known about how the LPMO-catalyzed redox reactions and electron transfers function within the plant cell wall matrix during biological decay.During biomass conversion by fungi, many of the lignin-and carbohydrate-active redox enzymes are expressed simultaneously with hydrolytic enzymes 11,12 , which points to a possible interplay between these enzyme systems. LPMO-encoding genes are abundant in the genomes of biomass degrading and plant pathogenic fungi, and when grown on lignocellulosic material, LPMOs are among the most highly expressed proteins [13][14][15][16] . Notably, there is ample evidence that LPMOs enhance the power of the fungal degradative enzyme machinery 17,18 . Today, LPMOs are important components in industrial enzyme cocktails used for saccharification of cellulosic biofuel feedstocks 19,20 .LPMOs are copper enzymes 6 , which cycle between Cu (I) and Cu (II) to activate molecular oxygen. Kim et al. (2014) suggested a mechanism involving the formation of a copper-oxyl radical that abstracts a hydrogen and then hydroxylates the substrate via an oxygen-rebound mechanism 21 . The details of oxygen activation were further elaborated by X-ray absorption studies of the active site copper, leading to the conclusion that the initial oxygen species is a super oxide 22 . During in vivo conditions, the electron donor may be CDH, but microorganisms may also utilize other approaches for providing electron donors to oxidative reactions. Lignin is one of the main structural components in plants and has an electron configuration that provides a low barrier for electron transfer. There are indications that lignin may act as electron donor for LPMOs 18,23 , but substantial evidence is scarce.One-electron transfer from lignin, proceeding via an outer sphere mechanism 24 , is well known and has been described for lignin oxidizing enzymes such as laccases 25 . The transfer may take place through direct interactions

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Lytic polysaccharide monooxygenases play a pivotal role in enzymatic deconstruction of plant cell wall material due to their ability to catalyze oxidative cleavage of glycosidic bonds. LPMOs may release different products, often in small amounts, with various oxidation patterns (C1 or C4) and with varying stabilities, making accurate analysis of product profiles a major challenge. So far, HPAEC has been the method of choice but it has limitations with respect to analysis of C4-oxidized products. Here, we compare various HPLC methods and present procedures that allow efficient separation of intact C1- and C4-oxidized products. We demonstrate that both PGC and HILIC (in WAX-mode) can separate C1- and C4-oxidized products and that PGC gives superior chromatographic performance. In contrast to HPAEC, these methods are directly compatible with mass spectroscopy and charged aerosol detection (CAD), which enables online peak validation and quantification with LOD levels in the low ng range. While the novel methods show lower resolution than HPAEC, this is compensated by easy peak identification, allowing, for example, discrimination between chromatographically highly similar native and C4-oxidized cello-oligomers. HPAEC-MS studies revealed chemical oxidation of C4-geminal diol products, which implies that peaks commonly believed to be C4-oxidized cello-oligomers, in fact are on-column generated derivatives. Non-destructive separation of C4-oxidized cello-oligosaccharides on the PGC column allowed us, for the first time, to isolate C4-oxidized standards. HPAEC fractionation of a purified C4-oxidized tetramer revealed that on-column decomposition leads to formation of the native trimer, which may explain why product mixtures generated by C4-oxidizing LPMOs seem to be rich in native oligosaccharides when analyzed by HPAEC. The findings and methods described here will aid in future studies in the emerging LPMO field.