Xylohexaose

A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan.[Pubmed:38589536]

Planta. 2024 Apr 8;259(5):115.

A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan. Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the Xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

Biochemical characterization of rice xylan biosynthetic enzymes in determining xylan chain elongation and substitutions.[Pubmed:38501734]

Plant Cell Physiol. 2024 Mar 19:pcae028.

Grass xylan consists of a linear chain of beta-1,4-linked xylosyl residues that often form domains substituted only with either arabinofuranose (Araf) or (methyl)glucuronic acid [(Me)GlcA] residues and it lacks the unique reducing end tetrasaccharide sequence found in dicot xylan. The mechanism of how grass xylan backbone elongation is initiated and how its distinctive substitution pattern is determined remain elusive. Here, we performed biochemical characterization of rice xylan biosynthetic enzymes, including xylan synthases, glucuronyltransferases and methyltransferases. Activity assays of rice xylan synthases demonstrated that they required short xylooligomers as acceptors for their activities. While rice xylan glucuronyltransferases effectively glucuronidated unsubstituted Xylohexaose acceptors, they transferred little GlcA residues onto Araf-substituted Xylohexaoses and rice xylan 3-O-arabinosyltransferase could not arabinosylate GlcA-substituted Xylohexaoses, indicating that their intrinsic biochemical properties may contribute to the distinctive substitution pattern of rice xylan. In addition, we found that rice xylan methyltransferase exhibited a low substrate binding affinity, which may explain the partial GlcA methylation in rice xylan. Furthermore, immunolocalization of xylan in xylem cells of both rice and Arabidopsis showed that it was deposited together with cellulose in secondary walls without forming xylan-rich nanodomains. Together, our findings provide new insights into the biochemical mechanisms underlying xylan backbone elongation and substitutions in grass species.

Quantifying CBM-Carbohydrate Interactions Using Microscale Thermophoresis.[Pubmed:37149525]

Methods Mol Biol. 2023;2657:103-114.

Microscale thermophoresis (MST) is an emerging technology for studying a broad range of biomolecular interactions with a high sensitivity. The affinity constant can be obtained for a wide range of molecules within minutes based on reactions in microliters. Here we describe the application of MST in quantifying protein-carbohydrate interactions. A CBM3a and a CBM4 are titrated with insoluble substrate (cellulose nanocrystal) and soluble oligosaccharide (Xylohexaose), respectively.

Discovery of a bifunctional xylanolytic enzyme with arabinoxylan arabinofuranohydrolase-d3 and endo-xylanase activities and its application in the hydrolysis of cereal arabinoxylans.[Pubmed:37096984]

Microb Biotechnol. 2023 Jul;16(7):1536-1547.

Xylanolytic enzymes, with both endo-xylanase and arabinoxylan arabinofuranohydrolase (AXH) activities, are attractive for the economically feasible conversion of recalcitrant arabinoxylan. However, their characterization and utilization of these enzymes in biotechnological applications have been limited. Here, we characterize a novel bifunctional enzyme, rAbf43A, cloned from a bacterial consortium that exhibits AXH and endo-xylanase activities. Hydrolytic pattern analyses revealed that the AXH activity belongs to AXHd3 because it attacked only the C(O)-3-linked arabinofuranosyl residues of double-substituted xylopyranosyl units of arabinoxylan and arabinoxylan-derived oligosaccharides, which are usually resistant to hydrolysis. The enzyme rAbf43A also liberated a series of xylo-oligosaccharides (XOSs) from beechwood xylan, Xylohexaose and xylopentaose, indicating that rAbf43A exhibited endo-xylanase activity. Homology modelling based on AlphaFold2 and site-directed mutagenesis identified three non-catalytic residues (H161, A270 and L505) located in the substrate-binding pocket essential for its dual-functionality, while the mutation of A117 located in the -1 subsite to the proline residue only affected its endo-xylanase activity. Additionally, rAbf43A showed significant synergistic action with the bifunctional xylanase/feruloyl esterase rXyn10A/Fae1A from the same bacterial consortium on insoluble wheat arabinoxylan and de-starched wheat bran degradation. When rXyn10A/Fae1A was added to the rAbf43A pre-hydrolyzed reactions, the amount of released reducing sugars, xylose and ferulic acid increased by 9.43% and 25.16%, 189.37% and 93.54%, 31.39% and 32.30%, respectively, in comparison with the sum of hydrolysis products released by each enzyme alone. The unique characteristics of rAbf43A position it as a promising candidate not only for designing high-performance enzyme cocktails but also for investigating the structure-function relationship of GH43 multifunctional enzymes.

Heterologous expression of family GH11 Aspergillus niger xylanase B (AnXylB11) in Pichia pastoris and competitive inhibition by riceXIP: An experimental and simulation study.[Pubmed:36252538]

Colloids Surf B Biointerfaces. 2022 Dec;220:112907.

The family GH11 Aspergillus niger JL15 xylanase B (AnXylB11) was heterologously expressed in Pichia pastoris X33. The recombinant AnXylB11 (reAnXylB11) was secreted into the culture medium with a molecular weight of approximately 33.0 kDa. The optimal temperature and pH of reAnXylB11 were 40 ℃ and 5.0, respectively. reAnXylB11 released xylobiose (X2)-Xylohexaose (X6) from beechwood xylan, with xylotriose (X3) as the primary product. The hydrolysates showed significant antioxidant activity. reAnXylB11 was also competitively inhibited by recombinant rice xylanase inhibitory protein (rePriceXIP), with an inhibition constant (K(i)) of 106.9 nM. Molecular dynamics (MD) simulations, non-covalent interactions (NCI), and binding free energy calculation and decomposition were conducted to decipher the interactional features between riceXIP and AnXyB11. Representative conformation of riceXIP-AnXylB11 complex showed that a U-shaped long loop between alpha(4) and beta(5) (K143-L152) of riceXIP was protruded into the catalytic groove and formed tight interaction with many key residues of AnXylB11. The binding free energy of riceXIP-AnXylB11 was calculated to be - 46.1 +/- 10.5 kcal/mol, with Coulomb and van der Waals forces contributing the most. NCI analysis showed that the hydrogen bonding networks such as R148(riceXIP)-E98(AnXyl11B), K143(riceXIP)-D138(AnXyl11B) and R148(riceXIP)-E189(AnXyl11B) provided terrific contributions to the interface interaction. The Laplacian of electron density values of atom pairs R148(riceXIP)@ 2HH1-E89(AnXylB11)@OE2 and N142(riceXIP)@ 1HD2-D138(AnXylB11)@OD1 were 0.12190 and 0.16009 a.u., respectively. Exploring the interactional features between xylanase and inhibitor protein may aid in constructing mutant xylanase that is insensitive to xylanase inhibitory proteins (XIs).

A novel reducing-end xylose-releasing exo-oligoxylanase (PphRex8A) from Paenibacillus physcomitrellae XB.[Pubmed:35785629]

Enzyme Microb Technol. 2022 Oct;160:110086.

In order to better understand the function of a putative GH8 xylanase gene in the xylan-degrading bacterium Paenibacillus physcomitrellae XB, a novel reducing-end xylose-releasing exo-oligoxylanase (Rex) PphRex8A of GH8 family was characterized. Phylogenetic analysis showed that it was clustered tightly with other published GH8 Rexs and exhibited the highest amino acid sequence identity (77.4 %) with the Rex of PbRex8 from P. barengoltzii G22. The three-dimensional (3D) structure of PphRex8A was also built based on the template of PbRex8 (5YXT) and three conserved catalytic active sites (Glu71, Asp263, and Asp129) were predicted and further confirmed by the enzymatic inactivity of their mutants (E71A, D129A, and D263A). The hydrolysis assay of PphRex8A showed that it could hydrolyze xylo-oligosaccharides (XOSs) with a degree of polymerization (DP) >/= 3, such as xylotriose (X3) through Xylohexaose (X6), and some natural XOSs, such as corncob xylan (CCX) and oat spelt xylan (OSX) to release xylose. However, it could not hydrolyze p-nitrophenyl-beta-D-xylopyranoside (pNPX), which suggested that it mainly released xylose from the reducing-end of XOSs and belonged to a Rex. In addition, PphRex8A also could deconstruct xylans with high DP, such as wheat arabinoxylan (AX) and beech wood xylan (BWX) to produce XOSs with DP3-6. Moreover, PphRex8A had synergistic effects with other xylanolytic enzymes of P. physcomitrellae XB, such as with PphXyn10 or PphXyn11 at a ratio of 1:3, or with PphXyl43B as a ratio of 3:1, significantly increasing the amounts of reducing sugars toward different xylan substrates. Thus, PphRex8A could be an exo-xylanase toward XOSs and could improve the deconstruction capability of high DP xylans, thereby complementing other xylanolytic enzymes to contribute to xylan degradation and improve the efficiency of converting hemicellulose biomass into energy by P. physcomitrellae XB.

A GH115 alpha-glucuronidase structure reveals dimerization-mediated substrate binding and a proton wire potentially important for catalysis.[Pubmed:35503213]

Acta Crystallogr D Struct Biol. 2022 May 1;78(Pt 5):658-668.

Xylan is a major constituent of plant cell walls and is a potential source of biomaterials, and the derived oligosaccharides have been shown to have prebiotic effects. Xylans can be highly substituted with different sugar moieties, which pose steric hindrance to the xylanases that catalyse the hydrolysis of the xylan backbone. One such substituent is alpha-D-glucuronic acid, which is linked to the O2' position of the beta-1,4 D-xylopyranoses composing the main chain of xylans. The xylan-specific alpha-glucuronidases from glycoside hydrolase family 115 (GH115) specifically catalyse the removal of alpha-D-glucuronic acid (GlcA) or methylated GlcA (MeGlcA). Here, the molecular basis by which the bacterial GH115 member wtsAgu115A interacts with the main chain of xylan and the indirect involvement of divalent ions in the formation of the Michaelis-Menten complex are described. A crystal structure at 2.65 A resolution of wtsAgu115A originating from a metagenome from an anaerobic digester fed with wastewater treatment sludge was determined in complex with Xylohexaose, and Asp303 was identified as the likely general acid. The residue acting as the general base could not be identified. However, a proton wire connecting the active site to the metal site was observed and hence a previous hypothesis suggesting a Grotthuss-like mechanism cannot be rejected. Only a single molecule was found in the asymmetric unit. However, wtsAgu115A forms a dimer with a symmetry-related molecule in the crystal lattice. The Xylohexaose moieties of the Xylohexaose are recognized by residues from both protomers, thus creating a Xylohexaose recognition site at the dimer interface. The dimer was confirmed by analytical size-exclusion chromatography in solution. Kinetic analysis with aldouronic acids resulted in a Hill coefficient of greater than 2, suggesting cooperativity between the two binding sites. Three Ca(2+) ions were identified in the wtsAgu115A structures. One Ca(2+) ion is of particular interest as it is coordinated by the residues of the loops that also interact with the substrate. Activity studies showed that the presence of Mg(2+) or Mn(2+) resulted in a higher activity towards aldouronic acids, while the less restrictive coordination geometry of Ca(2+) resulted in a decrease in activity.

Functional analysis of GT61 glycosyltransferases from grass species in xylan substitutions.[Pubmed:34821996]

Planta. 2021 Nov 25;254(6):131.

Multiple rice GT61 members were demonstrated to be xylan arabinosyltransferases (XATs) mediating 3-O-arabinosylation of xylan and the functions of XATs and xylan 2-O-xylosyltransferases were shown to be conserved in grass species. Xylan is the major hemicellulose in the cell walls of grass species and it is typified by having arabinofuranosyl (Araf) substitutions. In this report, we demonstrated that four previously uncharacterized, Golgi-localized glycosyltransferases residing in clade A or B of the rice GT61 family were able to mediate 3-O-arabinosylation of xylan when heterologously expressed in the Arabidopsis gux1/2/3 triple mutant. Biochemical characterization of their recombinant proteins established that they were xylan arabinosyltransferases (XATs) capable of transferring Araf residues onto Xylohexaose acceptors, and thus they were named OsXAT4, OsXAT5, OsXAT6 and OsXAT7. OsXAT5 and the previously identified OsXAT2 were shown to be able to arabinosylate xylooligomers with a degree of polymerization of as low as 3. Furthermore, a number of XAT homologs from maize, sorghum, Brachypodium and switchgrass were found to exhibit activities catalyzing Araf transfer onto Xylohexaose, indicating that they are XATs involved in xylan arabinosylation in these grass species. Moreover, we revealed that homologs of another GT61 member, xylan 2-O-xylosyltransferase (XYXT1), from these grass species could mediate 2-O-xylosylation of xylan when expressed in the Arabidopsis gux1/2/3 mutant. Together, our findings indicate that multiple OsXATs are involved in 3-O-arabinosylation of xylan and the functions of XATs and XYXTs are conserved in grass species.

Contributions and characteristics of two bifunctional GH43 beta-xylosidase /alpha-L-arabinofuranosidases with different structures on the xylan degradation of Paenibacillus physcomitrellae strain XB.[Pubmed:34687975]

Microbiol Res. 2021 Dec;253:126886.

Xylan is one of the major polymeric hemicellulosic constituents of lignocellulosic biomass, and its effective utilization by microorganisms is crucial for the economical production of biofuels. In this study, Paenibacillus physcomitrellae XB exhibited different xylan degradation ability on different substrates of corncob xylan (CCX), oat spelt xylan (OSX), wheat flour arabinoxylan (AX) and beech wood xylan (BWX). The RT-QPCR result showed that two genes (Pph_0602 and Pph_2344) belonging to the glycoside hydrolase family 43 were up-regulated more than 5-fold on CCX and xylose. Substrate-specific assays with purified proteins Ppxyl43A (Pph_0602) and Ppxyl43B (Pph_2344) revealed that both exhibited beta-xylosidase activity toward the chromogenic substrate p-nitrophenyl-beta-D-xylopyranoside, and alpha-L-arabinofuranosidase activity toward p-nitrophenyl-alpha-L-arabinofuranoside, indicating their bifunctionality. By testing their degradation characteristics on different natural substrates, it was found that both Ppxyl43A and Ppxyl43B showed similar degradation ability on CCX and OSX. Both enzymes could hydrolyze Xylohexaose and xylobiose completely to xylose, but could not hydrolyze BWX and AX, suggesting they mainly hydrolyze xylo-oligosaccharides by beta-xylosidase activity. Further analysis showed that both of them displayed very high pH stability and thermostability on the beta-xylosidase activity, but Ppxy143B exhibited wider pH and temperature ranges, higher pH and temperature stability, was less influenced by metal ions, and had a slower start-up response than Ppxyl43A. Given their predicted structure, it is likely that the enzymatic differences between Ppxyl43A and Ppxyl43B might be related to the extra C-terminus domain (GH43_C2) in Ppxyl43B, which could enhance the enzymatic stability while restricting the substrates' or metal ions' access to the active sites of Ppxyl43B. In conclusion, both Ppxyl43A and Ppxyl43B were beta-xylosidase/alpha-L-arabinofuranosidase bifunctional enzymes and might be useful in xylan biomass conversion, especially in the hydrolysis of xylo-oligosaccharides into xylose.

A Novel Multifunctional Arabinofuranosidase/Endoxylanase/beta-Xylosidase GH43 Enzyme from Paenibacillus curdlanolyticus B-6 and Its Synergistic Action To Produce Arabinose and Xylose from Cereal Arabinoxylan.[Pubmed:34613758]

Appl Environ Microbiol. 2021 Nov 24;87(24):e0173021.

PcAxy43B is a modular protein comprising a catalytic domain of glycoside hydrolase family 43 (GH43), a family 6 carbohydrate-binding module (CBM6), and a family 36 carbohydrate-binding module (CBM36) and found to be a novel multifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6. This enzyme exhibited alpha-l-arabinofuranosidase, endoxylanase, and beta-d-xylosidase activities. The alpha-l-arabinofuranosidase activity of PcAxy43B revealed a new property of GH43, via the release of both long-chain cereal arabinoxylan and short-chain arabinoxylooligosaccharide (AXOS), as well as release from both the C(O)(2) and C(O)(3) positions of AXOS, which is different from what has been seen for other arabinofuranosidases. PcAxy43B liberated a series of xylooligosaccharides (XOSs) from birchwood xylan and Xylohexaose, indicating that PcAxy43B exhibited endoxylanase activity. PcAxy43B produced xylose from xylobiose and reacted with p-nitrophenyl-beta-d-xylopyranoside as a result of beta-xylosidase activity. PcAxy43B effectively released arabinose together with XOSs and xylose from the highly arabinosyl-substituted rye arabinoxylan. Moreover, PcAxy43B showed significant synergistic action with the trifunctional endoxylanase/beta-xylosidase/alpha-l-arabinofuranosidase PcAxy43A and the endoxylanase Xyn10C from strain B-6, in which almost all products produced from rye arabinoxylan by these combined enzymes were arabinose and xylose. In addition, the presence of CBM36 was found to be necessary for the endoxylanase property of PcAxy43B. PcAxy43B is capable of hydrolyzing untreated cereal biomass, corn hull, and rice straw into XOSs and xylose. Hence, PcAxy43B, a significant accessory multifunctional xylanolytic enzyme, is a potential candidate for application in the saccharification of cereal biomass. IMPORTANCE Enzymatic saccharification of cereal biomass is a strategy for the production of fermented sugars from low-price raw materials. In the present study, PcAxy43B from P. curdlanolyticus B-6 was found to be a novel multifunctional alpha-l-arabinofuranosidase/endoxylanase/beta-d-xylosidase enzyme of glycoside hydrolase family 43. It is effective in releasing arabinose, xylose, and XOSs from the highly arabinosyl-substituted rye arabinoxylan, which is usually resistant to hydrolysis by xylanolytic enzymes. Moreover, almost all products produced from rye arabinoxylan by the combination of PcAxy43B with the trifunctional xylanolytic enzyme PcAxy43A and the endoxylanase Xyn10C from strain B-6 were arabinose and xylose, which can be used to produce several value-added products. In addition, PcAxy43B is capable of hydrolyzing untreated cereal biomass into XOSs and xylose. Thus, PcAxy43B is an important multifunctional xylanolytic enzyme with high potential in biotechnology.

A Comparative Study to Decipher the Structural and Dynamics Determinants Underlying the Activity and Thermal Stability of GH-11 Xylanases.[Pubmed:34073139]

Int J Mol Sci. 2021 May 31;22(11):5961.

With the growing need for renewable sources of energy, the interest for enzymes capable of biomass degradation has been increasing. In this paper, we consider two different xylanases from the GH-11 family: the particularly active GH-11 xylanase from Neocallimastix patriciarum, NpXyn11A, and the hyper-thermostable mutant of the environmentally isolated GH-11 xylanase, EvXyn11(TS). Our aim is to identify the molecular determinants underlying the enhanced capacities of these two enzymes to ultimately graft the abilities of one on the other. Molecular dynamics simulations of the respective free-enzymes and enzyme-Xylohexaose complexes were carried out at temperatures of 300, 340, and 500 K. An in-depth analysis of these MD simulations showed how differences in dynamics influence the activity and stability of these two enzymes and allowed us to study and understand in greater depth the molecular and structural basis of these two systems. In light of the results presented in this paper, the thumb region and the larger substrate binding cleft of NpXyn11A seem to play a major role on the activity of this enzyme. Its lower thermal stability may instead be caused by the higher flexibility of certain regions located further from the active site. Regions such as the N-ter, the loops located in the fingers region, the palm loop, and the helix loop seem to be less stable than in the hyper-thermostable EvXyn11(TS). By identifying molecular regions that are critical for the stability of these enzymes, this study allowed us to identify promising targets for engineering GH-11 xylanases. Eventually, we identify NpXyn11A as the ideal host for grafting the thermostabilizing traits of EvXyn11(TS).

Substrate Specificities of GH8, GH39, and GH52 beta-xylosidases from Bacillus halodurans C-125 Toward Substituted Xylooligosaccharides.[Pubmed:33394289]

Appl Biochem Biotechnol. 2021 Apr;193(4):1042-1055.

Substrate specificities of glycoside hydrolase families 8 (Rex), 39 (BhXyl39), and 52 (BhXyl52) beta-xylosidases from Bacillus halodurans C-125 were investigated. BhXyl39 hydrolyzed xylotriose most efficiently among the linear xylooligosaccharides. The activity decreased in the order of Xylohexaose > xylopentaose > xylotetraose and it had little effect on xylobiose. In contrast, BhXyl52 hydrolyzed xylobiose and xylotriose most efficiently, and its activity decreased when the main chain became longer as follows: xylotetraose > xylopentaose > Xylohexaose. Rex produced O-beta-D-xylopyranosyl-(1 --> 4)-[O-alpha-L-arabinofuranosyl-(1 --> 3)]-O-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranose (Ara(2)Xyl(3)) and O-beta-D-xylopyranosyl-(1 --> 4)-[O-4-O-methyl-alpha-D-glucuronopyranosyl-(l --> 2)]-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranose (MeGlcA(2)Xyl(3)), which lost a xylose residue from the reducing end of O-beta-D-xylopyranosyl-(1 --> 4)-[O-alpha-L-arabinofuranosyl-(1 --> 3)]-O-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranose (Ara(3)Xyl(4)) and O-beta-D-xylopyranosyl-(1 --> 4)-[O-4-O-methyl-alpha-D-glucuronopyranosyl-(1 --> 2)]-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranosyl-(1 --> 4)-beta-D-xylopyranose (MeGlcA(3)Xyl(4)). It was considered that there is no space to accommodate side chains at subsite -1. BhXyl39 rapidly hydrolyzes the non-reducing-end xylose linkages of MeGlcA(3)Xyl(4), while the arabinose branch does not significantly affect the enzyme activity because it degrades Ara(3)Xyl(4) as rapidly as unmodified xylotetraose. The model structure suggested that BhXyl39 enhanced the activity for MeGlcA(3)Xyl(4) by forming a hydrogen bond between glucuronic acid and Lys265. BhXyl52 did not hydrolyze Ara(3)Xyl(4) and MeGlcA(3)Xyl(4) because it has a narrow substrate binding pocket and 2- and 3-hydroxyl groups of xylose at subsite +1 hydrogen bond to the enzyme.

Effect of Oligosaccharide Degree of Polymerization on the Induction of Xylan-Degrading Enzymes by Fusarium oxysporum f. sp. Lycopersici.[Pubmed:33322262]

Molecules. 2020 Dec 11;25(24):5849.

Xylan is one of the most abundant carbohydrates on Earth. Complete degradation of xylan is achieved by the collaborative action of endo-beta-1,4-xylanases and beta-d-xylosidases and a number of accessories enzymes. In filamentous fungi, the xylanolytic system is controlled through induction and repression. However, the exact mechanism remains unclear. Substrates containing xylan promote the induction of xylanases, which release xylooligosaccharides. These, in turn, induce expression of xylanase-encoding genes. Here, we aimed to determine which xylan degradation products acted as inducers, and whether the size of the released oligomer correlated with its induction strength. To this end, we compared xylanase production by different inducers, such as sophorose, lactose, cellooligosaccharides, and xylooligosaccharides in Fusarium oxysporum f. sp. lycopersici. Results indicate that xylooligosaccharides are more effective than other substrates at inducing endoxylanase and beta-xylosidases. Moreover, we report a correlation between the degree of xylooligosaccharide polymerization and induction efficiency of each enzyme. Specifically, xylotetraose is the best inducer of endoxylanase, Xylohexaose of extracellular beta-xylosidase, and xylobiose of cell-bound beta-xylosidase.

Effects of oligolignol sizes and binding modes on a GH11 xylanase inhibition revealed by molecular modeling techniques.[Pubmed:32388588]

J Mol Model. 2020 May 9;26(6):124.

Lignin and phenolic compounds have been shown as the main recalcitrance for biomass decomposition, as they inhibit a number of lignocellulose-degrading enzymes. Understanding the inhibition mechanisms and energetic competitions with the native substrate is essential for the development of lignin resistive enzymes. In this study, atomistic detail of the size-dependent effects and binding modes of monomeric coniferyl alcohol, dimeric oligolignol, and tetrameric oligolignol made from coniferyl alcohols on the GH11 xylanase from Bacillus firmus strain K-1 was investigated by using molecular docking and atomistic molecular dynamics (MD) simulations. From the MD simulation results on the docked conformation of oligolignol binding within the "Cleft" and the "N-terminal," changes were observed both for protein conformations and positional binding of ligands, as binding with "Thumb" regions was found for all oligolignin models. Moreover, the uniquely stable "N-terminal" binding of the coniferyl alcohol monomer had no effect on the highly fluctuated Thumb region, showing no sign of inhibitory effect, and was in good agreement with recent studies. However, the inhibitory effect of oligolignols was size dependent, as the estimated binding energy of the tetrameric oligolignol became stronger than that of the Xylohexaose substrate, and the important binding residues were identified for future protein engineering attempts to enhance the lignin resistivity of GH11. Graphical Abstract Size-dependent binding modes of coniferyl alcohol monomers (upper panels) and the dimers (lower panels). Uniquely stable "N-terminal" binding of the monomer is shown to have no effect on the binding pocket, and hence no sign of inhibition, which was in good agreement with some recent studies.

A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities.[Pubmed:31980921]

Appl Microbiol Biotechnol. 2020 Mar;104(5):2079-2096.

PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the beta-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and Xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.