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ß-mannans and xylans are important components of the plant cell wall and they are acetylated to be protected from degradation by glycoside hydrolases. ß-mannans are widely present in human and animal diets as fiber from leguminous plants and as thickeners and stabilizers in processed foods. There are many fully characterized acetylxylan esterases (AcXEs); however, the enzymes deacetylating mannans are less understood. Here we present two carbohydrate esterases, RiCE2 and RiCE17, from the Firmicute Roseburia intestinalis, which together deacetylate complex galactoglucomannan (GGM). The three-dimensional (3D) structure of RiCE17 with a mannopentaose in the active site shows that the CBM35 domain of RiCE17 forms a confined complex, where the axially oriented C2-hydroxyl of a mannose residue points toward the Ser41 of the catalytic triad. Cavities on the RiCE17 surface may accept galactosylations at the C6 positions of mannose adjacent to the mannose residue being deacetylated (subsite -1 and +1). In-depth characterization of the two enzymes using time-resolved NMR, high-performance liquid chromatography (HPLC), and mass spectrometry demonstrates that they work in a complementary manner. RiCE17 exclusively removes the axially oriented 2-O-acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the RiCE2 is active on 3-O-, 4-O-, and 6-O-acetylations. Activity of RiCE2 is dependent on RiCE17 removing 2-O-acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2-O-specific RiCE17 provided insight into how temperature and pH affects acetyl migration on manno-oligosaccharides. [ABSTRACT FROM AUTHOR]

Pectic homogalacturonan ( HG) is one of the main constituents of plant cell walls. When processed to low degrees of esterification, HG can form complexes with divalent calcium ions. These macromolecular structures (also called egg boxes) play an important role in determining the biomechanics of cell walls and in mediating cell-to-cell adhesion. Current immunological methods enable only steady-state detection of egg box formation in situ. Here we present a tool for efficient real-time visualisation of available sites for HG crosslinking within cell wall microdomains. Our approach is based on calcium-mediated binding of fluorescently tagged long oligogalacturonides ( OGs) with endogenous de-esterified HG. We established that more than seven galacturonic acid residues in the HG chain are required to form a stable complex with endogenous HG through calcium complexation in situ, confirming a recently suggested thermodynamic model. Using defined carbohydrate microarrays, we show that the long OG probe binds exclusively to HG that has a very low degree of esterification and in the presence of divalent ions. We used this probe to study real-time dynamics of HG during elongation of Arabidopsis pollen tubes and root hairs. Our results suggest a different spatial organisation of incorporation and processing of HG in the cell walls of these two tip-growing structures. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases ( LPMOs) are important for the enzymatic conversion of biomass and seem to play a key role in degradation of the plant cell wall. In this study, we characterize an LPMO from the fungal plant pathogen Fusarium graminearum ( Fg LPMO9A) that catalyzes the mixed C1/C4 oxidative cleavage of cellulose and xyloglucan, but is inactive toward other (1,4)-linked β-glucans. Our findings indicate that Fg LPMO9A has unprecedented broad specificity on xyloglucan, cleaving any glycosidic bond in the β-glucan main chain, regardless of xylosyl substitutions. Interestingly, we found that when incubated with a mixture of xyloglucan and cellulose, Fg LPMO9A efficiently attacks the xyloglucan, whereas cellulose conversion is inhibited. This suggests that removal of hemicellulose may be the true function of this LPMO during biomass conversion. [ABSTRACT FROM AUTHOR]

Background: Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes. Results: We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained. Conclusions: We show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems. [ABSTRACT FROM AUTHOR]

Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus Neurospora crassa, NcLPMO9A (NCU02240), NcLPMO9C (NCU02916), and NcLPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound NcLPMO9A at 1.6-Å resolution and found that NcLPMO9A and NcLPMO9C, containing a CBM1 carbohydrate-binding module, bind cellulose more strongly and were less susceptible to inactivation than NcLPMO9D, which lacks a CBM. All three LPMOs were active on tamarind xyloglucan and konjac glucomannan, generating similar products but clearly differing in activity levels. Importantly, in some cases, the addition of phosphoric acid-swollen cellulose (PASC) had a major effect on activity: NcLPMO9A was active on xyloglucan only in the presence of PASC, and PASC enhanced NcLPMO9D activity on glucomannan. Interestingly, the three enzymes also exhibited large differences in their interactions with enzymatic electron donors, which could reflect that they are optimized to act with different reducing partners. All three enzymes efficiently used H2O2 as a cosubstrate, yielding product profiles identical to those obtained in O2-driven reactions with PASC, xyloglucan, or glucomannan. Our results indicate that seemingly similar LPMOs act preferentially on different types of copolymeric substructures in the plant cell wall, possibly because these LPMOs are functionally adapted to distinct niches differing in the types of available reductants. [ABSTRACT FROM AUTHOR]

Highlights: [•] Xylo-oligos’ released during pretreatment have DP≤20 (most 3–8); more acetylated in stems. [•] AX and MLG released during pretreatment; xylan, XG, glucan and mannan remain only alkali-extractable. [•] All polysaccharides partly digestible after pretreatment, regardless extractability. [•] Digestibility correlates with polysaccharides’ structural organisation. [•] Results correlate with previous models of the primary type II cell wall. [Copyright &y& Elsevier]