Xylan Binding Module Research Articles

Molecular insight into a new low-affinity xylan binding module from the xylanolytic gut symbiont Roseburiaintestinalis.

Efficient capture of glycans, the prime metabolic resources in the human gut, confers a key competitive advantage for gut microbiota members equipped with extracellular glycoside hydrolases (GHs) to target these substrates. The association of glycans to the bacterial cell surface is typically mediated by carbohydrate binding modules (CBMs). Here, we report the structure of RiCBM86 appended to a GH family 10 xylanase from Roseburiaintestinalis. This CBM represents a new family of xylan binding CBMs present in xylanases from abundant and prevalent healthy human gut Clostridiales. RiCBM86 adopts a canonical β-sandwich fold, but shows structural divergence from known CBMs. The structure of RiCBM86 has been determined with a bound xylohexaose, which revealed an open and shallow binding site. RiCBM86 recognizes only a single xylosyl ring with direct hydrogen bonds. This mode of recognition is unprecedented amongst previously reported xylan binding type-B CBMs that display more extensive hydrogen-bonding patterns to their ligands or employ Ca2+ to mediate ligand-binding. The architecture of RiCBM86 is consistent with an atypically low binding affinity (KD about0.5mm for xylohexaose) compared to most xylan binding CBMs. Analyses using NMR spectroscopy corroborated the observations from the complex structure and the preference of RiCBM86 to arabinoxylan over glucuronoxylan, consistent with the largely negatively charged surface flanking the binding site. Mutational analysis and affinity electrophoresis established the importance of key binding residues, which are conserved in the family. This study provides novel insight into the structural features that shape low-affinity CBMs that mediate extended bacterial glycan capture in the human gut niche. DATABASES: Structural data are available in the protein data bank database under the accession number 6SGF. Sequence data are available in the GenBank database under the accession number EEV01588.1. The assignment of the Roseburiaintestinalis xylan binding module into the CBM86 new family is available in the CAZy database (http://www.cazy.org/CBM86.html).

Fusion of a xylan-binding module to gluco-oligosaccharide oxidase increases activity and promotes stable immobilization.

The xylan-binding module Clostridium thermocellum CBM22A was successfully fused to a gluco-oligosaccharide oxidase, GOOX-VN, from Sarocladium strictum via a short TP linker, allowing the fused protein to effectively bind different xylans. The presence of the CtCBM22A at the N-terminal of GOOX-VN increased catalytic activity on mono- and oligo-saccharides by 2-3 fold while not affecting binding affinity to these substrates. Notably, both GOOX-VN and its CBM fusion also showed oxidation of xylo-oligosaccharides with degrees of polymerization greater than six. Whereas fusion to CtCBM22A did not alter the thermostability of GOOX-VN or reduce substrate inhibition, CtCBM22A_GOOX-VN could be immobilized to insoluble oat spelt xylan while retaining wild-type activity. QCM-D analysis showed that the fused enzyme remained bound during oxidation. These features could be harnessed to generate hemicellulose-based biosensors that detect and quantify the presence of different oligosaccharides.

A new bi-modular endo-β-1,4-xylanase KRICT PX-3 from whole genome sequence of Paenibacillus terrae HPL-003

A new bi-modular, wide pH spectrum and highly active xylanase KRICT PX3 (JF320814) isolated from Paenibacillus terrae HPL-003 (KCTC11987BP) has been cloned and expressed in Escherichia coli. Purified recombinant xylanase KRICT PX-3 (1,620bp, 540aa, NCBI accession number JF320814) showed highly active at 55°C in pH 4.0–11.0, and stability for at least 24h at 50°C, and exhibited Km and Vmax of 0.2mg/mL and 153.8U/mg on birchwood xylan. Most common ions did not affect the enzyme activity at 1mM concentration. This enzyme could belong to glycoside hydrolase family 10 because hydrolyzed glucuronoxylan and arabinoxylan substrate to xylobiose, xylotriose, and some traces of xylose as hydrolysis products. Model 3-D structure was composed of two domains, the catalytic domain of a (β/α)8 barrel fold while the small domain probably functions as a xylan binding domain, and the two domains are connected by a flexible linker peptide (PPLAIEKDIPSL). However, sequence alignment between xylan-binding module in this xylanase KRICT PX3 and CBM22 showed 21% of identity and 35% of similarity. This xylanase structure showed a distinctive group of enzyme cluster separately from the rest of GH10 xylanases, and seems to constitute a new type of xylanases.

The carbohydrate-binding module of xylanase from Nonomuraea flexuosa decreases its non-productive adsorption on lignin

BackgroundThe enzymatic hydrolysis step converting lignocellulosic materials into fermentable sugars is recognized as one of the major limiting steps in biomass-to-ethanol process due to the low efficiency of enzymes and their cost. Xylanases have been found to be important in the improvement of the hydrolysis of cellulose due to the close interaction of cellulose and xylan. In this work, the effects of carbohydrate-binding module (CBM family II) of the xylanase 11 from Nonomuraea flexuosa (Nf Xyn11) on the adsorption and hydrolytic efficiency toward isolated xylan and lignocellulosic materials were investigated.ResultsThe intact family 11 xylanase of N. flexuosa clearly adsorbed on wheat straw and lignin, following the Langmuir-type isotherm. The presence of the CBM in the xylanase increased the adsorption and hydrolytic efficiency on insoluble oat spelt xylan. But the presence of the CBM did not increase adsorption on pretreated wheat straw or isolated lignin. On the contrary, the CBM decreased the adsorption of the core protein to lignin containing substrates, indicating that the CBM of N. flexuosa xylanase did not contribute to the non-productive adsorption.ConclusionThe CBM of the N. flexuosa xylanase was shown to be a xylan-binding module, which had low affinity on cellulose. The CBM of the N. flexuosa xylanase reduced the non-specific adsorption of the core protein to lignin and showed potential for improving the hydrolysis of lignocellulosic materials to platform sugars.

Functional assembly and characterization of a modular xylanosome for hemicellulose hydrolysis in yeast

Five trimeric xylanosomes were successfully assembled on the cell surface of Saccharomyces cerevisiae. Three dockerin-tagged fungal enzymes, an endoxylanase (XynAc) from Thermomyces lanuginosus, a β-xylosidase (XlnDt) from Aspergillus niger and an acetylxylan esterase (AwAXEf) from Aspergillus awamori, were displayed for the synergistic saccharification of birchwood xylan. The surface-expression scaffoldins were modular constructs with or without carbohydrate binding modules from Thermotoga maritima (family 22) or Clostridium thermocellum (family 3). The synergy due to enzyme-enzyme and enzyme-substrate proximity, and the effects of binding domain choice and position on xylan hydrolysis were determined. The scaffoldin-based enzymes (with no binding domain) showed a 1.6-fold increase in hydrolytic activity over free enzymes; this can be attributed to enzyme-enzyme proximity within the scaffoldin. The addition of a xylan binding domain from T. maritima improved hydrolysis by 2.1-fold relative to the scaffoldin without a binding domain (signifying enzyme-substrate synergy), and 3.3-fold over free enzymes, with a xylose productivity of 105 mg g(-1) substrate after 72 h hydrolysis. This system was also superior to the xylanosome carrying the cellulose binding module from C. thermocellum by 1.4-fold. Furthermore, swapping the xylan binding module position within the scaffoldin resulted in 1.5-fold more hydrolysis when the binding domain was adjacent to the endoxylanase. These results demonstrate the applicability of designer xylanosomes toward hemicellulose saccharification in yeast, and the importance of the choice and position of the carbohydrate binding module for enhanced synergy.

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and approximately 1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.

Substrate Recognition of a Family 10 Xylanase from Streptomyces olivaceoviridis E-86: A Study by Site-directed Mutagenesis to Make an Hindrance around the Entrance toward the Substrate-binding Cleft

A family 10 xylanase of Streptomyces olivaceoviridis E-86 (SoXyn10A) is known to have a modular structure where an N-terminal catalytic module and a C-terminal xylan-binding module (XBM) are connected by a Gly/Pro-rich linker. The crystal structure of native SoXyn10A indicates that the XBM is located beyond -3 subsite from the substrate binding cleft. To investigate the mechanism of xylan binding by SoXyn10A, several kinds of cleft mutants of SoXyn10A were constructed. The mutants had an hindrance in the outer side of the (-) subsite or (+) subsite in the substrate binding cleft. Circular dichroism spectral analysis indicated that the mutant enzymes fold as a native enzyme. Kinetic studies for the mutants were performed by using p-nitrophenyl-β-D-xylobioside. The kcat/Km value did not change for the mutants which had a hindrance in the outer side of the (+) subsite while the value significantly decreased for the mutants which had a hindrance in the outer side of the (-) subsite, suggesting that xylan come into the substrate binding cleft of SoXyn10A from the direction of (-) subsites. The activities of the mutants against insoluble xylan were tested with or without XBM. All the mutants possessing XBM showed higher activity than those possessing the catalytic modules without XBM, indicating that the linker sequence connecting the catalytic module and XBM is flexible so that XBM binds to insoluble xylan to increase the concentration around the enzyme.

Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.

The First Thermodynamic Characterization of β-1,3-Xylanase from a Marine Bacterium

Sequence analysis of beta-1,3-xylanase (TxyA) from a marine bacterium, Alcaligenes sp. strain XY-234 implied that an xylan-binding module belonging to carbohydrate-binding module family 31 (TxyA-CBM) is separated from a catalytic module belonging to glycosyl hydrolase family 26 (TxyA-CM) by a putative glycine-rich linker [Okazaki, F., et al. (2002) J. Bacteriol. 184: 2399-2403]. In order to reveal the role of these structural features of TxyA, two modules, TxyA-CBM and TxyA-CM, were constructed, and those modules and full-length TxyA were characterized by thermodynamic studies. TxyA-CBM and TxyA-CM showed full reversible folding from denaturant-induced unfolded forms, exhibited higher thermodynamic stabilities. The conformational stability of both truncated modules is industrially desirable, as well as aiding the understanding of the enzymatic characterization of the two modules of beta-1,3-xylanase separated by a long linker.

Preparation and preliminary X-ray analysis of the catalytic module of beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4.

Beta-1,3-xylanase (1,3-beta-D-xylan xylanohydrolase; EC 3.2.1.32) is an enzyme capable of hydrolyzing beta-1,3-xylan. The newly cloned beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 (XYL4) exhibited a modular structure consisting of three modules: an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two C-terminal xylan-binding modules belonging to carbohydrate-binding module family 31. Despite substantial crystallization screening, crystallization of the recombinant XYL4 was not accomplished. However, the deletion mutant of XYL4, composed of a catalytic module without a xylan-binding module, was crystallized. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 51.6, b = 75.8, c = 82.0 A. X-ray diffraction data were collected to 1.44 A resolution.

The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase.

The solution structure is presented for the second family 4 carbohydrate binding module (CBM4-2) of xylanase 10A from the thermophilic bacterium Rhodothermus marinus. CBM4-2, which binds xylan tightly, has a beta-sandwich structure formed by 11 strands, and contains a prominent cleft. From NMR titrations, it is shown that the cleft is the binding site for xylan, and that the main amino acids interacting with xylan are Asn31, Tyr69, Glu72, Phe110, Arg115, and His146. Key liganding residues are Tyr69 and Phe110, which form stacking interactions with the sugar. It is suggested that the loops on which the rings are displayed can alter their conformation on substrate binding, which may have functional importance. Comparison both with other family 4 cellulose binding modules and with the structurally similar family 22 xylan binding module shows that the key aromatic residues are in similar positions, and that the bottom of the cleft is much more hydrophobic in the cellulose binding modules than the xylan binding proteins. It is concluded that substrate specificity is determined by a combination of ring orientation and the nature of the residues lining the bottom of the binding cleft.

74. Development and evaluation of a PCR to detect Mycoplasma putrefaciens directly from goats

A new bi-modular, wide pH spectrum and highly active xylanase KRICT PX3 (JF320814) isolated from Paenibacillus terrae HPL-003 (KCTC11987BP) has been cloned and expressed in Escherichia coli. Purified recombinant xylanase KRICT PX-3 (1,620 bp, 540aa, NCBI accession number JF320814) showed highly active at 55 °C in pH 4.0–11.0, and stability for at least 24 h at 50 °C, and exhibited Km and Vmax of 0.2 mg/mL and 153.8 U/mg on birchwood xylan. Most common ions did not affect the enzyme activity at 1 mM concentration. This enzyme could belong to glycoside hydrolase family 10 because hydrolyzed glucuronoxylan and arabinoxylan substrate to xylobiose, xylotriose, and some traces of xylose as hydrolysis products. Model 3-D structure was composed of two domains, the catalytic domain of a (β/α)8 barrel fold while the small domain probably functions as a xylan binding domain, and the two domains are connected by a flexible linker peptide (PPLAIEKDIPSL). However, sequence alignment between xylan-binding module in this xylanase KRICT PX3 and CBM22 showed 21% of identity and 35% of similarity. This xylanase structure showed a distinctive group of enzyme cluster separately from the rest of GH10 xylanases, and seems to constitute a new type of xylanases.

1H and 15N chemical shift assignments of the C-terminal xylan binding module of C. fimi xylanase 11A

1H and 15N chemical shift assignments of the C-terminal xylan binding module of C. fimi xylanase 11A

Role of hydrogen bonding in the interaction between a xylan binding module and xylan.

NMR studies of the internal family 2b carbohydrate binding module (CBM2b-1) of Cellulomonas fimi xylanase 11A have identified six polar residues and two aromatic residues that interact with its target ligand, xylan. To investigate the importance of the various interactions, free energy and enthalpy changes have been measured for the binding of xylan to native and mutant forms of CBM2b-1. The data show that the two aromatic residues, Trp 259 and Trp 291, play a critical role in the binding, and similarly that mutants N264A and T316A have no affinity for the xylose polymer. Interestingly, mutations E257A, Q288A, N292A, E257A/Q288A, E257A/N292A, and E257A/N292A/Q288A do not significantly diminish the affinity of CBM2b-1 for the xylose polymers, but do influence the thermodynamics driving the protein-carbohydrate interactions. These thermodynamic parameters have been interpreted in light of a fresh understanding of enthalpy-entropy compensation and show the following. (1) For proteins whose ligands are bound on an exposed surface, hydrogen bonding confers little specificity or affinity. It also displays little cooperativity. Most specificity and affinity derive from binding between the face of sugar rings and aromatic rings. (2) Loss of hydrogen bonding interactions leads to a redistribution of the remaining bonding interactions such that the entropic mobility of the ligand is maximized, at the expense (if necessary) of enthalpically favorable bonds. (3) Changes in entropy and enthalpy in the binding between polysaccharide and a range of mutants can be interpreted by considering changes in binding and flexibility, without any need to consider solvent reorganization.

The Structural Basis for the Ligand Specificity of Family 2 Carbohydrate-binding Modules

The interactions of proteins with polysaccharides play a key role in the microbial hydrolysis of cellulose and xylan, the most abundant organic molecules in the biosphere, and are thus pivotal to the recycling of photosynthetically fixed carbon. Enzymes that attack these recalcitrant polymers have a modular structure comprising catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). The largest prokaryotic CBM family, CBM2, contains members that bind cellulose (CBM2a) and xylan (CBM2b), respectively. A possible explanation for the different ligand specificity of CBM2b is that one of the surface tryptophans involved in the protein-carbohydrate interaction is rotated by 90 degrees compared with its position in CBM2a (thus matching the structure of the binding site to the helical secondary structure of xylan), which may be promoted by a single amino acid difference between the two families. Here we show that by mutation of this single residue (Arg-262-->Gly), a CBM2b xylan-binding module completely loses its affinity for xylan and becomes a cellulose-binding module. The structural effect of the mutation has been revealed using NMR spectroscopy, which confirms that Trp-259 rotates 90 degrees to lie flat against the protein surface. Except for this one residue, the mutation only results in minor changes to the structure. The mutated protein interacts with cellulose using the same residues that the wild-type CBM2b uses to interact with xylan, suggesting that the recognition is of the secondary structure of the polysaccharide rather than any specific recognition of the absence or presence of functional groups.

Phage display of xylan-binding module of xylanase J from alkaliphilic Bacillus sp. strain 41M-1.

Xylanase J from alkaliphilic Bacillus sp. strain 41M-1 has a family 11/G catalytic domain and a xylan-binding module (XBM). The XBM of xylanase J was displayed on the surface of filamentous bacteriophage. The XBM expressed on the phage surface retained binding activity to xylan. Random mutations were introduced in the XBM gene by error-prone PCR, and the repertoire was cloned for display on phage. Sequence analysis of the xylan-binding activity-deficient mutants revealed that Phe 284 and Trp317 of the XBM would contribute to the xylan-binding activity.