Lytic polysaccharide monooxygenases:a crystallographer's view on a new class of biomass-degrading enzymes

Lytic polysaccharide monooxygenases (LPMOs) are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future

Oligosaccharide Binding and Thermostability of Two Related AA9 Lytic Polysaccharide Monooxygenases

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

Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the Nε2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His