A family 10 Thermoascus aurantiacus xylanase utilizes arabinose decorations of xylan as significant substrate specificity determinants

Xylan, which is a key component of the plant cell wall, consists of a backbone of beta-1,4-linked xylose residues that are decorated with arabinofuranose, acetyl, 4-O-methyl d-glucuronic acid and ferulate. The backbone of xylan is hydrolysed by endo-beta1,4-xylanases (xylanases); however, it is unclear whether the various side-chains of the polysaccharide are utilized by these enzymes as significant substrate specificity determinants. To address this question we have determined the crystal structure of a family 10 xylanase from Thermoascus aurantiacus, in complex with xylobiose containing an arabinofuranosyl-ferulate side-chain. We show that the distal glycone subsite of the enzyme makes extensive direct and indirect interactions with the arabinose side-chain, while the ferulate moiety is solvent-exposed. Consistent with the 3D structural data, the xylanase displays fourfold more activity against xylotriose in which the non-reducing moiety is linked to an arabinose side-chain, compared to the undecorated form of the oligosacchairde. These data indicate that the sugar decorations of xylans in the T.aurantiacus family 10 xylanase, rather than simply being accommodated, can be significant substrate specificity determinants.     

Structural and biochemical analysis of Cellvibrio japonicus xylanase 10C: how variation in substrate-binding cleft influences the catalytic profile of family GH-10 xylanases

Microbial degradation of the plant cell wall is the primary mechanism by which carbon is utilized in the biosphere. The hydrolysis of xylan, by endo-beta-1,4-xylanases (xylanases), is one of the key reactions in this process. Although amino acid sequence variations are evident in the substrate binding cleft of "family GH10" xylanases (see afmb.cnrs-mrs.fr/CAZY/), their biochemical significance is unclear. The Cellvibrio japonicus GH10 xylanase CjXyn10C is a bi-modular enzyme comprising a GH10 catalytic module and a family 15 carbohydrate-binding module. The three-dimensional structure at 1.85 A, presented here, shows that the sequence joining the two modules is disordered, confirming that linker sequences in modular glycoside hydrolases are highly flexible. CjXyn10C hydrolyzes xylan at a rate similar to other previously described GH10 enzymes but displays very low activity against xylooligosaccharides. The poor activity on short substrates reflects weak binding at the -2 subsite of the enzyme. Comparison of CjXyn10C with other family GH10 enzymes reveals "polymorphisms" in the substrate binding cleft including a glutamate/glycine substitution at the -2 subsite and a tyrosine insertion in the -2/-3 glycone region of the substrate binding cleft, both of which contribute to the unusual properties of the enzyme. The CjXyn10C-substrate complex shows that Tyr-340 stacks against the xylose residue located at the -3 subsite, and the properties of Y340A support the view that this tyrosine plays a pivotal role in substrate binding at this location. The generic importance of using CjXyn10C as a template in predicting the biochemical properties of GH10 xylanases is discussed.     

Mode of action of endo-beta-1,4-xylanases of families 10 and 11 on acidic xylooligosaccharides

Mode of action of endo-beta-1,4-xylanases (EXs) of glycoside hydrolase families 10 (GH-10) and 11 (GH-11) was examined on various acidic xylooligosaccharides. As expected, none of the enzymes of GH-10 cleaved aldotetraouronic acid (MeGlcA3Xyl3), which is the shortest acidic product of the action of these EXs on glucuronoxylan. Surprisingly, aldopentaouronic acid (MeGlcA3Xyl4) was also not attacked. Only aldohexaouronic acid (MeGlcA3Xyl5) served as a substrate and was cleaved to xylobiose and aldotetraouronic acid. These results suggested that binding of xylopyranosyl residue in the -2 subsite is prerequisite for cleavage of the linkage adjacent to the xylopyranosyl unit carrying MeGlcA. EXs of family GH-11 cleaved neither aldotetraouronic acid, nor aldopentaouronic acid, which is in agreement with their action on glucuronoxylan. Aldohexaouronic acid was cleaved to aldopentaouronic acid and xylobiose without any production of xylose, suggesting that a xylosyl transfer reaction is involved in the degradation of the substrate by EXs of GH-11.     

Purification and some properties of high-molecular-weight xylanases, the xylanases 4 and 5 of Aeromonas caviae W-61

Aeromonas caviae W-61 produces multiple extracellular xylanases, the xylanases 1, 2, 3, 4, and 5 [Nguyen, V. D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993)]. Here we purified and characterized high-molecular-weight xylanases, the xylanases 4 and 5 from the culture fluids of the bacterium. The purified xylanases 4 and 5, which had molecular masses of 120 and 140 kDa, respectively, were endo-beta-1,4-xylanases with similar enzymatic properties except for trans-xylosidase activity. The xylanase 4 showed a prominent transxylosidase activity when xylotriose and xylotetraose were used as the substrates, while the xylanase 5 had little transxylosidase activity under the same conditions. Protein sequencing indicated that the xylanase 4 was a C-terminally-truncated xylanase 5, suggesting that the C-terminal truncation of the xylanase 5 may endow the enzyme with transxylosidase activity.     

Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases

Endo-β1,4-xylanases (xylanases) hydrolyse the β1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown. Furthermore, the mechanism for the differences in the inhibition of fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these issues we report the crystal structure and biochemical properties of the Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high catalytic activity and is one of the few fungal GH11 proteins not inhibited by XIP-I. Although the structure of NpXyn11A could not be determined in complex with substrates, we have been able to investigate how GH11 enzymes hydrolyse decorated substrates by solving the crystal structure of a second GH11 xylanase, EnXyn11A (encoded by an environmental DNA sample), bound to ferulic acid-1,5-arabinofuranose-α1,3-xylotriose (FAX₃). The crystal structure of the EnXyn11A-FAX₃ complex shows that solvent exposure of the backbone xylose O2 and O3 groups at subsites −3 and +2 allow accommodation of α1,2-linked 4-methyl-D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic interactions at the +2 subsite, indicating that the decoration may represent a specificity determinant at this aglycone subsite. The structure of NpXyn11A reveals potential −3 and +3 subsites that are kinetically significant. The extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases, may explain why the Neocallimastix enzyme displays unusually high catalytic activity. Finally, the crystal structure of NpXyn11A shows that the resistance of the enzyme to XIP-I is not due solely to insertions in the loop connecting β strands 11 and 12, as suggested previously, but is highly complex.     

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.     

The mechanisms by which family 10 glycoside hydrolases bind decorated substrates

Endo-beta-1,4-xylanases (xylanases), which cleave beta-1,4 glycosidic bonds in the xylan backbone, are important components of the repertoire of enzymes that catalyze plant cell wall degradation. The mechanism by which these enzymes are able to hydrolyze a range of decorated xylans remains unclear. Here we reveal the three-dimensional structure, determined by x-ray crystallography, and the catalytic properties of the Cellvibrio mixtus enzyme Xyn10B (CmXyn10B), the most active GH10 xylanase described to date. The crystal structure of the enzyme in complex with xylopentaose reveals that at the +1 subsite the xylose moiety is sandwiched between hydrophobic residues, which is likely to mediate tighter binding than in other GH10 xylanases. The crystal structure of the xylanase in complex with a range of decorated xylooligosaccharides reveals how this enzyme is able to hydrolyze substituted xylan. Solvent exposure of the O-2 groups of xylose at the +4, +3, +1, and -3 subsites may allow accommodation of the alpha-1,2-linked 4-O-methyl-d-glucuronic acid side chain in glucuronoxylan at these locations. Furthermore, the uronic acid makes hydrogen bonds and hydrophobic interactions with the enzyme at the +1 subsite, indicating that the sugar decorations in glucuronoxylan are targeted to this proximal aglycone binding site. Accommodation of 3'-linked l-arabinofuranoside decorations is observed in the -2 subsite and could, most likely, be tolerated when bound to xylosides in -3 and +4. A notable feature of the binding mode of decorated substrates is the way in which the subsite specificities are tailored both to prevent the formation of "dead-end" reaction products and to facilitate synergy with the xylan degradation-accessory enzymes such as alpha-glucuronidase. The data described in this report and in the accompanying paper indicate that the complementarity in the binding of decorated substrates between the glycone and aglycone regions appears to be a conserved feature of GH10 xylanases.     

Secondary structure and NMR assignments of Bacillus circulans xylanase.

Bacillus circulans xylanase (BCX) is a member of the family of low molecular weight endo-beta-(1,4)-xylanases. The main-chain 1H, 13C, and 15N resonances of this 20.4-kDa enzyme were assigned using heteronuclear NMR experiments recorded on a combination of selectively and uniformly labeled protein samples. Using chemical shift, NOE, J coupling, and amide hydrogen exchange information, 14 beta-strands, arranged in a network of three beta-sheets, and a single alpha-helix were identified in BCX. The NMR-derived secondary structure and beta-sheet topology agree closely with that observed in the crystal structure of this protein. The HN of Ile 118 has a strongly upfield-shifted resonance at 4.03 ppm, indicative of a potential amide-aromatic hydrogen bond to the indole ring of Trp 71. This interaction, which is conserved in all low molecular weight xylanases of known structure, may play an important role in establishing the active site conformation of these enzymes. Following hen egg white and bacteriophage T4 lysozymes, B. circulans xylanase represents the third family of beta-glycanases for which extensive NMR assignments have been reported. These assignments provide the background for detailed studies of the mechanism of carbohydrate recognition and hydrolysis by this bacterial xylanase.     

An alternative approach for the synthesis of fluorogenic substrates of endo-beta-(1-->4)-xylanases and some applications

Fluorogenic substrates of endo-beta-(1-->4)-xylanases (EXs), 4-methylumbelliferyl beta-glycosides of xylobiose and xylotriose were synthesized from fully acetylated oligosaccharides using the alpha-trichloroacetimidate procedure. A commercially available syrup containing xylose and xylo-oligosaccharides was used as the starting material. Both fluorogenic glycosides were found to be suitable substrates for EXs, particularly for sensitive detection of the enzymes in electrophoretic gels and their in situ localization on sections of fruiting bodies of some plants, such as tomato, potato and eggplant, all of the family Solanaceae.     

The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site

Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 A. The protein displays a beta-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and beta-1,3-1,4-mixed linked glucans.     

Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome

Cereals contain proteinaceous inhibitors of endo-beta-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography followed by affinity chromatography, were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI- and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity. Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.     

Production and Properties of Extracellular Endoxylanase from Neurospora crassa

Neurospora crassa 870 produced 14 and 0.025 U of extracellular xylanase (1,4-beta-d-xylan xylanohydrolase; EC 3.2.1.8) and beta-xylosidase (1,4-beta-xylan xylohydrolase; EC 3.2.1.37) per ml, respectively, in 4 days when commercial xylan was used as a carbon source. The effects of pH and carbon sources on xylanase production by N. crassa are discussed. Two xylanases (I and II) were purified and had pI values of 4.8 and 4.5 and molecular weights of 33,000 and 30,000. The maximum degree of hydrolysis of xylan by the extracellular culture broth was 66% in 4 h. The end products of xylan hydrolysis by xylanase I and II showed the presence of xylose, xylobiose, xylotriose, xylotetraose, xylopentose, and arabinose, indicating that they are endoxylanases capable of hydrolyzing 1,3-alpha-l-arabinofuranosyl branch points. Both xylanases showed activity toward carboxymethyl cellulose but no activity toward para-nitrophenyl-beta-d-xyloside or laminarin. Xylanase I showed appreciable activity toward para-nitrophenyl-beta-d-glucoside, whereas xylanase II was inactive.     

On the Modes of Action of Three Xylanases Produced by a Strain of Aspergillus niger van Tieghem

The modes of action of three xylanases (I, II and III) produced by Aspergillus niger van Tieghem on several substrates were investigated. Xylanase I possesed the strongest activity against xylooligosaccharides among the three enzymes and converted them into xylose and xylobiose. Xylanase II and III catalyzed a glycosylating reaction and produced higher polymerized xylooligosaccharides from xylotetraose or xylopentaose. Among three enzymes, xylanase II could split α1,3-arabinofuranosidic bond of arabinose-xylose mixed oligosaccharides.

In the case of hydrolysis by three xylanases on xylan and arabinoxylan, the maximum hydrolysis degree and the reaction products were compared with each other. From the results, some speculation were made concerning the modes of action of the enzymes.     

Substrate and product hydrolysis specificity in family 11 glycoside hydrolases: an analysis of <Emphasis Type="Italic">Penicillium funiculosum</Emphasis> and <Emphasis Type="Italic">Penicillium griseofulvum</Emphasis> xylanases

Two genes encoding family 11 endo-(1,4)-β-xylanases from Penicillium griseofulvum (PgXynA) and Penicillium funiculosum (PfXynC) were heterologously expressed in Escherichia coli as glutathione S-transferase fusion proteins, and the recombinant enzymes were purified after affinity chromatography and proteolysis. PgXynA and PfXynC were identical to their native counterparts in terms of molecular mass, pI, N-terminal sequence, optimum pH, and enzymatic activity towards arabinoxylan. Further investigation of the rate and pattern of hydrolysis of PgXynA and PfXynC on wheat soluble arabinoxylan showed the predominant production of xylotriose and xylobiose as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing chain length (n) of oligomer up to n = 6, suggesting that the specificity region of both Penicillium xylanases spans about six xylose units. In contrast to PfXynC, PgXynA was found insensitive to the wheat xylanase inhibitor protein XIP-I.     

Characterization of the biochemical properties of recombinant Xyn10C from a marine bacterium, <Emphasis Type="Italic">Saccharophagus degradans</Emphasis> 2-40

Endo-1,4-β-xylanases are mostly classified into glycoside hydrolase (GH) family 10 or 11. In this study, we examined the catalytic functions of a recombinant endo-1,4-β-xylanase belonging to GH10 (Xyn10C) from a marine bacterium, Saccharophagus degradans 2-40. Optimal activity of this enzyme was evident at 30 °C and pH 7.0, but activity remained even at low temperatures, indicating its adaptation to cold. With respect to other xylanases known to be active in cold temperatures, Xyn10C is unique in that it showed maximal activity in the presence of 2 M of NaCl. The action patterns of recombinant Xyn10C on xylans from hardwood and softwood differed in part, but the enzyme hydrolyzed polysaccharidic substrates primarily to xylobiose and xylotriose through xylo-oligosaccharides, releasing a small amount of xylose. The K _m and V _max values on birchwood xylan were 10.4 mg mL^?1 and 253 µmol mg^?1 min^?1, respectively. The efficient catalytic function of Xyn10C on short-length xylo-oligosaccharide chains was similar to the typical function of other known GH10 xylanases.     

Purification,characterization and mass spectrometric identification of two thermophilic xylanases from Sporotrichum thermophile

Two xylanases were purified to electrophoretic homogeneity from the thermophilic fungus Sporotrichum thermophile grown in a submerged liquid culture using wheat straw as carbon source. The enzymes, StXyn1 and StXyn2, have molecular masses of 24 kDa and 48 kDa, respectively, and are optimally active at pH 5 and at 60 °C. Both enzymes displayed remarkable stability up to 50 °C for 1 h, exhibiting a half-life of 60 min (StXyn1) and 115 min (StXyn2) at 60 °C. Biochemical characterization of the two xylanases against poly- and oligosaccharides indicated that StXyn1 and StXyn2 hydrolytic profiles match those of xylanase family 11 and family 10, respectively. LC–MS/MS analysis provided peptide mass and sequence information that assisted the identification of the corresponding xylanase genes from the S. thermophile genome and the classification of the two purified StXyn1 and StXyn2 as a family GH11 and GH10 endo-1,4-β-xylanases, respectively.     

The endo-beta-1,4-xylanase xyn11A is required for virulence in Botrytis cinerea

Phytopathogenic fungi can degrade xylan, an abundant hemicellulose in plant cell walls, by the coordinate action of a group of extracellular enzymes. Among these, endo-beta-1,4-xylanases carry out the initial breakdown by cleaving internal bonds in the polymer backbone. We have isolated and characterized a gene, xyn11A, coding for an endo-beta-1,4-xylanase belonging to family 11 of glycosyl hydrolases. xyn11A was shown to be induced by xylan and repressed by glucose and to be expressed in planta. The disruption of xyn11A caused only a moderate decrease, about 30%, in the level of extracellular endo-beta-1-4-xylanase activity and in the growth rate, with beechwood xylan as the only carbon source. However, deletion of the gene had a more pronounced effect on virulence, delaying the appearance of secondary lesions and reducing the average lesion size by more than 70%. Reintroducing the wild-type gene into the mutant strains reversed this phenotype back to wild type.     

Functional analysis of glycoside hydrolase family 8 xylanases shows narrow but distinct substrate specificities and biotechnological potential

The potential of glycoside hydrolase family (GH) 8 xylanases in biotechnological applications is virtually unexplored. Therefore, the substrate preference and hydrolysis product profiles of two GH8 xylanases were evaluated to investigate their activities and substrate specificities. A GH8 xylanase from an uncultured bacterium (rXyn8) shows endo action but very selectively releases xylotriose from its substrates. It has a higher activity than the Pseudoalteromonas haloplanktis GH8 endo-xylanase (PhXyl) on xylononaose and smaller xylo-oligosaccharides. PhXyl preferably degrades xylan substrates with a high degree of polymerization. It is sterically more hindered by arabinose substituents than rXyn8, producing larger end hydrolysis products. The specificities of rXyn8 and PhXyl differ completely from these of the previously described GH8 xylanases from Bifidobacterium adolescentis (BaRexA) and Bacillus halodurans (BhRex). As reducing-end xylose-releasing exo-oligoxylanases, they selectively release xylose from the reducing end of small xylo-oligosaccharides. The findings of this study show that GH8 xylanases have a narrow substrate specificity, but also one that strongly varies between family members and is distinct from that of GH10 and GH11 xylanases. Structural comparison of rXyn8, PhXyl, BaRexA, and BhRex showed that subtle amino acid changes in the glycon as well as the aglycon subsites probably form the basis of the observed differences between GH8 xylanases. GH8 xylanases, therefore, are an interesting group of enzymes, with potential towards engineering and applications.     

Fusarium graminearum xylanases show different functional stabilities,substrate specificities and inhibition sensitivities

When grown on arabinoxylan as the sole carbon source, the cereal phytopathogen Fusarium graminearum expresses four xylanases. Cloning and heterologous expression of the corresponding xylanase encoding genes and analysis of general biochemical properties, substrate specificities and inhibition sensitivities revealed some marked differences. XylA and XylB are glycoside hydrolase family (GH) 11 xylanases, while XylC and XylD belong to GH10. pH and temperature for optimal activity of the enzymes were between 6.0 and 7.0 and 40 °C, respectively. Interestingly, XylC displayed remarkable pH stability as it retained most of its activity even after pre-incubation at pH 1.0 and 13.0 for 120 min at room temperature. All xylanases hydrolysed xylotetraose, xylopentaose and xylohexaose, but to different extents, while only XylC and XylD hydrolysed xylotriose. The two GH10 xylanases released a higher percentage of smaller products from xylan and xylo-oligosaccharides than did their GH11 counterparts. Analysis of kinetic properties revealed that wheat arabinoxylan is the favoured XylC substrate while XylA and XylB prefer sparsely substituted oat spelt xylan. XylC and XylD were inhibited by xylanase inhibiting protein (XIP), while XylA and XylB were sensitive to Triticum aestivum xylanase inhibitor (TAXI). Because of its pH stability and preference for arabinoxylan, XylC is a valuable candidate for use in biotechnological applications.