Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation

Abstract

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

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

“…Because NcLPMO9C has been found to act on soluble substrates, including cellodextrins and xyloglucan fragments (13,14), attempts were made to obtain crystallographic data for an enzyme⅐substrate complex. Despite the use of a variety of substrates (thio-linked cello-oligosaccharides with a degree of polymerization of 2-5, thio-linked xylopentaose, cello-oligosaccharides with a degree of polymerization of 5 and 7, xyloglucooligosaccharides, low molecular lignin, and ␤-glucan) and a massive number of crystallization trials using both soaking and co-crystallization, such data were not obtained.…”

“…To analyze activity on polymeric substrates, we employed the fact that the C4-oxidizing NcLPMO9C generates novel reducing ends (14). 5 mg/ml xyloglucan from tamarind seed (Megazyme International) or PASC was incubated with 4 M copper-saturated NcLPMO9C or NcLPMO9C-N in 40 mM sodium phosphate, pH 6.5, and in the presence or absence of 2 mM ascorbic acid.…”

“…However, it was noted that the abundance, the large sequence variation, and the varying domain composition of LPMOs encoded in the genomes of biomass-degrading microorganisms suggest that some of these enzymes could have other substrates (1). Indeed, three studies published in 2014 have broadened the LPMO paradigm by demonstrating cleavage of soluble cellodextrins (13), hemicelluloses with ␤(134)-glucan backbones (14), and even starch (15). Based on sequence characteristics, LPMOs are currently categorized in auxiliary activity (AA) families 9 -11 and 13 of the CAZy database (16).…”

“…We have recently discovered that NcLPMO9C, a C4-oxidizing AA9 LPMO (hereafter LPMO9) from Neurospora crassa also known as NCU02916 or NcGH61-3, shows activity on soluble cellodextrins and hemicelluloses with ␤(134)-glucan backbones, including xyloglucan, glucomannan, and ␤-glucan (13,14). NcLPMO9C is a two-domain protein, containing an N-terminal catalytic AA9 domain and a family 1 carbohydratebinding module (CBM1), that is connected through a serineand threonine-rich linker comprising ϳ50 amino acid residues.…”

A family of starch-active polysaccharide monooxygenases

“…Double helices can form between amylose and amylopectin in starch, which further complicates the structure (23)(24)(25). Recently, a cellulose-active PMO from N. crassa (22) was found to act on soluble cellodextrins (40) and hemicelluloses that contain β(1→4) linked glucose and glucose derivative units (41). The crystalline surface has thus not been required for all previously identified PMOs; however, β(1→4) linkages of glucose and glucose derivatives have been essential.…”