Lytic polysaccharide monooxygenases (LPMOs) are industrially important copper-dependent enzymes that oxidatively cleave polysaccharides. Here we present a functional and structural characterization of two closely related AA9-family LPMOs from Lentinus similis (LsAA9A) and Collariella virescens (CvAA9A). LsAA9A and CvAA9A cleave a range of polysaccharides, including cellulose, xyloglucan, mixed-linkage glucan and glucomannan. LsAA9A additionally cleaves isolated xylan substrates. The structures of CvAA9A and of LsAA9A bound to cellulosic and non-cellulosic oligosaccharides provide insight into the molecular determinants of their specificity. Spectroscopic measurements reveal differences in copper co-ordination upon the binding of xylan and glucans. LsAA9A activity is less sensitive to the reducing agent potential when cleaving xylan, suggesting that distinct catalytic mechanisms exist for xylan and glucan cleavage. Overall, these data show that AA9 LPMOs can display different apparent substrate specificities dependent upon both productive protein–carbohydrate interactions across a binding surface and also electronic considerations at the copper active site.

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

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Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which cleave polysaccharide substrates oxidatively. First discovered because of their action on recalcitrant crystalline substrates (chitin and cellulose) a number of LPMOs are now reported to act on soluble substrates including oligosaccharides. However, crystallographic complexes with oligosaccharides have only been reported for a single LPMO so far, an enzyme from the basidiomycete fungus Lentinus similis (LsAA9_A). Here we present a more detailed comparative study of LsAA9_A and an LPMO from the ascomycete fungus Collariella virescens (CvAA9_A) with which it shares 41.5% sequence identity. LsAA9_A is considerably more thermostable than CvAA9_A, and the structural basis for the difference has been investigated. We have compared the patterns of oligosaccharide cleavage and the patterns of binding in several new crystal structures explaining the basis for product preferences by the two enzymes. Obtaining structural information on complexes of LPMOs with carbohydrates has proven very difficult in general judging from the structures reported in the literature thus far and this can only partly be attributed to low affinity for small substrates. We have thus evaluated the use of differential scanning fluorimetry as a guide to obtaining complex structures. Furthermore, an analysis of crystal packing of LPMOs and glycoside hydrolases, corroborates the hypothesis that active site occlusion is a very significant problem for LPMO-substrate interaction analysis by crystallography, due to their relatively flat and extended substrate binding sites.

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