The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.

Bonds broken and formed during the mixed-linkage glucan : xyloglucan endotransglucosylase reaction catalysed by Equisetum hetero-trans-ß-glucanase.

Mixed-linkage glucan∶xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-ß-glucanase (HTG), which among land plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-ß-d-glucan (MLG), with radioactive oligosaccharides of xyloglucan as the acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and gel-permeation chromatography (GPC). Equisetum HTG consistently cleaved the MLG at the third consecutive ß-(1→4)-bond following (towards the reducing terminus) a ß-(1→3)-bond. It then formed a ß-(1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its xyloglucan endotransglucosylase/hydrolase subfamily membership. Using size-homogeneous barley MLG as the donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative to the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.

Hetero-trans-ß-glucanase, an enzyme unique to Equisetum plants, functionalizes cellulose.

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that 'cut and paste' certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell-wall polysaccharides (xyloglucan, mannans, mixed-linkage ß-glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero-trans-ß-glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early-diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero-transglycosylation: its preferred donor substrates (cellulose or mixed-linkage ß-glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose-xyloglucan and mixed-linkage ß-glucan-xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter-linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better-known xyloglucan-acting homo-transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable 'green' technology for industrially manipulating biomass.

Probing the molecular architecture of Arabidopsis thaliana secondary cell walls using two- and three-dimensional (13)C solid state nuclear magnetic resonance spectroscopy.

The plant secondary cell wall is a thickened polysaccharide and phenolic structure, providing mechanical strength to cells, particularly in woody tissues. It is the main feedstock for the developing bioenergy and green chemistry industries. Despite the role that molecular architecture (the arrangement of biopolymers relative to each other, and their conformations) plays in dictating biomass properties, such as recalcitrance to breakdown, it is poorly understood. Here, unprocessed dry (13)C-labeled stems from the model plant Arabidopsis thaliana were analyzed by a variety of (13)C solid state magic angle spinning nuclear magnetic resonance methods, such as one-dimensional cross-polarization and direct polarization, two-dimensional refocused INADEQUATE, RFDR, PDSD, and three-dimensional DARR, demonstrating their viability for the study of native polymer arrangements in intact secondary cell walls. All carbon sites of the two main glucose environments in cellulose (previously assigned to microfibril surface and interior residues) are clearly resolved, as are carbon sites of the other major components of the secondary cell wall: xylan and lignin. The xylan carbon 4 chemical shift is markedly different from that reported previously for solution or primary cell wall xylan, indicating significant changes in the helical conformation in these dried stems. Furthermore, the shift span indicates that xylan adopts a wide range of conformations in this material, with very little in the 31 conformation typical of xylan in solution. Additionally, spatial connections of noncarbohydrate species were observed with both cellulose peaks conventionally assigned as "surface" and as "interior" cellulose environments, raising questions about the origin of these two cellulose signals.

Mixed-linkage glucan:xyloglucan endotransglucosylase (MXE) re-models hemicelluloses in Equisetum shoots but not in barley shoots or Equisetum callus.

Among land-plant hemicelluloses, xyloglucan is ubiquitous, whereas mixed-linkage (1→3),(1→4)-ß-D-glucan (MLG) is confined to the Poales (e.g. cereals) and Equisetales (horsetails). The enzyme MLG:xyloglucan endotransglucosylase (MXE) grafts MLG to xyloglucan. In Equisetum, MXE often exceeds extractable xyloglucan endotransglucosylase (XET) activity; curiously, cereals lack extractable MXE. We investigated whether barley possesses inextractable MXE. Grafting of endogenous MLG or xyloglucan onto exogenous [(3)H]xyloglucan oligosaccharides in vivo indicated MXE and XET action, respectively. Extractable MXE and XET activities were assayed in vitro. MXE and XET actions were both detectable in living Equisetum fluviatile shoots, the MXE : XET ratio increasing with age. However, only XET action was observed in barley coleoptiles, leaves and roots (which all contained MLG) and in E. fluviatile intercalary meristems and callus (which lacked MLG). In E. fluviatile, extractable MXE activity was high in mature shoots, but extremely low in callus and young shoots; in E. arvense strobili, it was undetectable. Barley possesses neither extractable nor inextractable MXE, despite containing both of its substrates and high XET activity. As the Poales are xyloglucan-poor, the role of their abundant endotransglucosylases remains enigmatic. The distribution of MXE action and activity within Equisetum suggests a strengthening role in ageing tissues.

Three highly acidic Equisetum XTHs differ from hetero-trans-ß-glucanase in donor substrate specificity and are predominantly xyloglucan homo-transglucosylases.

Transglycanases are enzymes that remodel the primary cell wall in plants, potentially loosening and/or strengthening it. Xyloglucan endotransglucosylase (XET; EC 2.4.1.207), ubiquitous in land plants, is a homo-transglucanase activity (donor, xyloglucan; acceptor, xyloglucan) exhibited by XTH (xyloglucan endotransglucosylase/hydrolase) proteins. By contrast, hetero-trans-ß-glucanase (HTG) is the only known enzyme that is preferentially a hetero-transglucanase. Its two main hetero-transglucanase activities are MLG : xyloglucan endotransglucosylase (MXE) and cellulose : xyloglucan endotransglucosylase (CXE). HTG is highly acidic and found only in the evolutionarily isolated genus of fern-allies, Equisetum. We now report genes for three new highly acidic HTG-related XTHs in E. fluviatile (EfXTH-A, EfXTH-H and EfXTH-I). We expressed them heterologously in Pichia and tested the encoded proteins' enzymic activities to determine whether their acidity and/or their Equisetum-specific sequences might confer high hetero-transglucanase activity. Untransformed Pichia was found to secrete MLG-degrading enzyme(s), which had to be removed for reliable MXE assays. All three acidic EfXTHs exhibited very predominantly XET activity, although low but measurable hetero-transglucanase activities (MXE and CXE) were also detected in EfXTH-H and EfXTH-I. We conclude that the extremely high hetero-transglucanase activities of Equisetum HTG are not emulated by similarly acidic Equisetum XTHs that share up to 55.5% sequence identity with HTG.

An even pattern of xylan substitution is critical for interaction with cellulose in plant cell walls.

Xylan and cellulose are abundant polysaccharides in vascular plants and essential for secondary cell wall strength. Acetate or glucuronic acid decorations are exclusively found on even-numbered residues in most of the glucuronoxylan polymer. It has been proposed that this even-specific positioning of the decorations might permit docking of xylan onto the hydrophilic face of a cellulose microfibril 1-3 . Consequently, xylan adopts a flattened ribbon-like twofold screw conformation when bound to cellulose in the cell wall 4 . Here we show that ESKIMO1/XOAT1/TBL29, a xylan-specific O-acetyltransferase, is necessary for generation of the even pattern of acetyl esters on xylan in Arabidopsis. The reduced acetylation in the esk1 mutant deregulates the position-specific activity of the xylan glucuronosyltransferase GUX1, and so the even pattern of glucuronic acid on the xylan is lost. Solid-state NMR of intact cell walls shows that, without the even-patterned xylan decorations, xylan does not interact normally with cellulose fibrils. We conclude that the even pattern of xylan substitutions seen across vascular plants enables the interaction of xylan with hydrophilic faces of cellulose fibrils, and is essential for development of normal plant secondary cell walls.

Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR.

Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall. 13C solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, supported by in silico predictions of chemical shifts, shows both two- and threefold screw xylan conformations are present in fresh Arabidopsis stems. The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.

Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by ß-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.

Considerations in the search for mixed-linkage (1→3),(1→4)-ß-D-glucan-active endotransglycosylases.

Xyloglucan endotransglucosylase, catalyzed by XTH subfamily members, is thought to play crucial roles in plant cell wall physiology. Recent discovery of endotransglycosylases active on other hemicelluloses extend our understanding of the physiological scope of endotransglycosylation in general. Discovery in Poaceaen XTHs of endotransglycosylases which act on Poaceaen-prevalent hemicelluloses, such as MLG, could reconcile the apparent incongruence between the large size of Poaceaen putative XTH families and the low xyloglucan content of their cell walls. Here, I speculate on hypothetical MLG-active endotransglycosylases and highlight potential hindrances to their discovery. It is suggested that because the location of ß-(1→3) bonds within MLG oligosaccharides (MLGOs) could define their ability to act as endotranglycosylase acceptor substrates: a) thorough probing of substrate specificities necessitates the use of MLGOs created using different endo-glycanases; and b) endogenous plant exo-glycosidases, which can hinder endotranglycosylase assays by degrading acceptor substrates, might prove particularly troublesome where MLGOs are concerned.

An unexpectedly lichenase-stable hexasaccharide from cereal, horsetail and lichen mixed-linkage ß-glucans (MLGs): implications for MLG subunit distribution.

Mixed-linkage (1→3),(1→4)-ß-d-glucan (MLG) is a biologically and technologically important hemicellulose, known to occur in three widely separated lineages: the Poales (including grasses and cereals), Equisetum (fern-allies), and some lichens e.g. Iceland moss (Cetraria islandica). Lichenase (E.C. 3.2.1.73) is widely assumed to hydrolyse all (1→4) bonds that immediately follow (1→3) bonds in MLG, generating predominantly the tetrasaccharide ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→3)-d-Glc (G4G4G3G; MLG4), the corresponding trisaccharide (G4G3G; MLG3), and sometimes also laminaribiose (G3G; MLG2). The ratio of the oligosaccharides produced characterises each polysaccharide. We report here that digestion of MLG from barley (Hordeum vulgare), Equisetum arvense and C. islandica by Bacillus subtilis lichenase also yields the unexpectedly stable hexasaccharide, ß-d-Glcp-(1→3)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→3)-d-Glc (G3G4G4G4G3G, i.e. MLG2-MLG4), identified by thin-layer chromatography, gel-permeation chromatography, HPLC (HPAEC), ß-glucosidase digestion, (1)H/(13)C-NMR spectroscopy and mass spectrometry. On HPLC, G3G4G4G4G3G is the major constituent of a peak previously ascribed solely to the nonasaccharide G4G4G4G4G4G4G4G3G. Because it was widely presumed that lichenase would cleave G3G4G4G4G3G to MLG2+MLG4, our data both redefine the substrate specificity of Bacillus lichenase and show previous attempts to characterise MLGs by HPLC of lichenase-digests to be flawed. MLG2 subunits are particularly underestimated; often reported as negligible, they are here shown to be an appreciable constituent of MLGs from all three lineages. We also show that there is no appreciable yield of water-soluble lichenase products with DP>9; potential identities of products previously labelled DP>9 are suggested. Finally, this discovery also provides a opportunity to investigate the spatial distribution of subunits along the MLG chain. We show that MLG2 subunits in barley and Cetraria MLG are not randomly distributed, but predominantly found at the non-reducing end of MLG4 subunits.