The efficient depolymerization of lignocellulosic biomass to fermentable sugars by enzymatic hydrolysis is a key step in the transition towards a more environmentally friendly and sustainable bio-economy. However, the complexity and recalcitrant nature of the substrate limit enzyme performance on lignocellulosic plant biomass, and at present the enzyme cocktails required for depolymerization represent a major cost in the production of biomass-based chemicals and fuels. The recent discovery of lytic polysaccharide monooxygenases (LPMOs) has changed our general understanding of polysaccharide deconstruction, and given rise to high expectations for further development of enzyme tools for biomass processing, since LPMOs enhance the activity of glycoside hydrolases.
LPMOs are copper-dependent enzymes that oxidize recalcitrant polysaccharides such as chitin and cellulose in the presence of dioxygen, and an external electron donor. Before the discovery of their enzymatic function, in 2010, LPMOs were classified as either family 33 of carbohydrate-binding modules (now family 10 of auxiliary activities, AA10, LPMO10) or family 61 of glycoside hydrolases (now AA9, LPMO9). Prior to the studies presented here, catalytic activity had just been demonstrated for a chitin-active bacterial AA10-type of LPMO from Serratia marcescens called CBP21. The work on CBP21 formed the basis for the first goal of this study, namely finding or engineering an LPMO targeting cellulose substrates. Paper I describes CelS2, a naturally occurring AA10-type LPMO from the Gram-positive bacterium Streptomyces coelicolor that cleaves crystalline cellulose and produces C1-oxidized cello-oligosaccharides appearing in solution as aldonic acids. The generation of oxidized products was demonstrated using both mass spectrometry and chromatographic methods. CelS2, which comprises an N-terminal AA10 and a Cterminal cellulose-binding carbohydrate-binding module classified as CBM2, represents the first described LPMO that is active on cellulose. It was shown that CelS2 stimulates the release of soluble sugars from filter paper by Celluclast® (a commercial enzyme cocktail).
Papers II and III of this study describe structure-function studies of cellulose-active AA10-type LPMOs with the purpose of unraveling the basic characteristics of these proteins and perhaps identify factors determining substrate specificity and the regioselectivity of hydroxylation (C1 versus C4 oxidation). Paper II describes a comparative study of four C1-oxidizing LPMOs, two of which are active on chitin and two on cellulose, and includes the description of one novel chitin-active LPMO10 (BlLPMO10A from Bacillus licheniformis) and one novel cellulose-active LPMO10 (E8 from Thermobifida fusca). Sequence analysis showed that all residues in the immediate copper coordination sphere were conserved in these four LPMOs. Conversely, electron paramagnetic resonance spectroscopy (EPR) analyses indicated that the electronic environments of the copper differed between the chitin- and cellulose-active LPMOs. The differences in the EPR spectra are thus likely to reflect variation in residues outside the direct copper coordination sphere, where the chitin-active and cellulose-active AA10-type of LPMOs indeed show considerable variation.
Paper III presents the first crystal structures of cellulose-active AA10-type LPMOs, which allowed for the first time a structural comparison of LPMOs with different substrate specificities. The two S. coelicolor LPMO for which the structures were determined, CelS2 and ScLPMO10B, represent a conserved pair of LPMOs found in cellulolytic actinomycetes. The two enzymes are pregulated during growth on cellulose substrates and we show that they act synergistically when degrading cellulose. CelS2 shows strict C1- oxidation on cellulose substrates, whereas ScLPMO10B catalyzes oxidation of C1 and C4 in cellulose, as well as C1-oxidation on β-chitin. A structural comparison of the two cellulose-active LPMO10s revealed a difference in the copper oordination sphere that may relate to the (in)ability to oxidize C4. Structural comparisons of chitin-active and celluloseactive LPMO10s revealed a potential binding-pocket for a C2 acetamido group in chitinactive LPMO10s only. All LPMO10s had similar redox potentials and copper binding affinities, but showed a substrate-dependent difference in EPR spectra, as discussed in Paper II. Substrate-specificity thus seems to be determined by variation in substrate-binding and –positioning combined with variation in the electronic structure of the copper site.
In conclusion, this study represents the discovery and first in-depth characterization of LPMOs from family 10 of auxiliary activities that are active on cellulose. The work presented here has provided fundamental insight into how these enzymes work and contributed to method development, thereby constituting an important basis for future LPMO research
