Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (<Emphasis Type="Italic">Ct</Emphasis>CBM11) of cellulosomal bifunctional cellulase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.     

Structure and functional investigation of ligand binding by a family 35 carbohydrate binding module (CtCBM35) of β-mannanase of family 26 glycoside hydrolase from Clostridium thermocellum

Functional attributes of recombinant CtCBM35 (family 35 carbohydrate binding module) of β-mannanase of family 26 Glycoside Hydrolase from Clostridium thermocellum were deduced by biochemical and in silico approaches. Ligand-binding analysis of expressed CtCBM35 analyzed by affinity-gel electrophoresis and fluorescence spectroscopy exhibited association constants K _a ～ 1.2·10⁵ and 3.0·10⁵ M^?1 with locust bean galactomannan and mannotriose, respectively. However, CtCBM35 showed low ligand-binding affinity with insoluble ivory nut mannan with K _a of 5.0·10^?5 M^?1. Unfolding transition analysis by fluorescence spectroscopy explained the conformational changes of CtCBM35 in the presence of guanidine hydrochloride (5 M) and urea (6.25 M). This explained that CtCBM35 has good conformational stability and requires higher free energy of denaturation to invoke unfolding. The three-dimensional (3-D) model of CtCBM35 from C. thermocellum generated by Modeller9v8 displayed predominance of β-sheets arranged as β-jelly-roll fold. The secondary structure of CtCBM35 by PredictProtein showed the presence of two α-helices (3%), 12 β-sheets (45%), and 15 random coils (52%). Secondary structural element analysis of cloned, expressed, and purified recombinant CtCBM35 by circular dichroism also corroborated the in silico predicted secondary structure. Multiple sequence alignment of CtCBM35 showed conserved residues (Tyr123, Gly124, and Phe125), which are commonly observed in mannan specific CBMs. Docking analysis of CtCBM35 with manno-oligosaccharide displayed the involvement of Tyr26, Gln29, Asn43, Trp66, Tyr68, Leu69, Arg76, and Leu127 residues, making polar contact with the ligand molecules. Ligand docking analysis of CtCBM35 exhibiting higher binding affinity with mannotriose and galactomannan (Man-Gal-Man moiety) substantiated the affinity binding and fluorescence results, displaying similar values of K _a.     

Fusion of a family 9 cellulose-binding module improves catalytic potential of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> cellodextrin phosphorylase on insoluble cellulose

Clostridium thermocellum cellodextrin phosphorylase (CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).     

Structural Analysis of Glucuronoxylan-specific Xyn30D and Its Attached CBM35 Domain Gives Insights into the Role of Modularity in Specificity

Glucuronoxylanase Xyn30D is a modular enzyme containing a family 30 glycoside hydrolase catalytic domain and an attached carbohydrate binding module of the CBM35 family. We present here the three-dimensional structure of the full-length Xyn30D at 2.4 Å resolution. The catalytic domain folds into an (α/β)₈ barrel with an associated β-structure, whereas the attached CBM35 displays a jellyroll β-sandwich including two calcium ions. Although both domains fold in an independent manner, the linker region makes polar interactions with the catalytic domain, allowing a moderate flexibility. The ancillary Xyn30D-CBM35 domain has been expressed and crystallized, and its binding abilities have been investigated by soaking experiments. Only glucuronic acid-containing ligands produced complexes, and their structures have been solved. A calcium-dependent glucuronic acid binding site shows distinctive structural features as compared with other uronic acid-specific CBM35s, because the presence of two aromatic residues delineates a wider pocket. The nonconserved Glu¹²⁹ makes a bidentate link to calcium and defines region E, previously identified as specificity hot spot. The molecular surface of Xyn30D-CBM35 shows a unique stretch of negative charge distribution extending from its binding pocket that might indicate some oriented interaction with its target substrate. The binding ability of Xyn30D-CBM35 to different xylans was analyzed by affinity gel electrophoresis. Some binding was observed with rye glucuronoarabinoxylan in presence of calcium chelating EDTA, which would indicate that Xyn30D-CBM35 might establish interaction to other components of xylan, such as arabinose decorations of glucuronoarabinoxylan. A role in depolymerization of highly substituted chemically complex xylans is proposed.     

Enhanced Polysaccharide Binding and Activity on Linear β-Glucans through Addition of Carbohydrate-Binding Modules to Either Terminus of a Glucooligosaccharide Oxidase

The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, β-(1→3,1→4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30 % and on insoluble crystalline as well as amorphous cellulose by over 50 %.     

The Unique Binding Mode of Cellulosomal CBM4 from Clostridium thermocellum Cellobiohydrolase A

The crystal structure of the carbohydrate-binding module (CBM) 4 Ig fused domain from the cellulosomal cellulase cellobiohydrolase A (CbhA) of Clostridium thermocellum was solved in complex with cellobiose at 2.11 Å resolution. This is the first cellulosomal CBM4 crystal structure reported to date. It is similar to the previously solved noncellulosomal soluble oligosaccharide-binding CBM4 structures. However, this new structure possesses a significant feature—a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common to all cellulosomal clostridial CBM4 modules. Our results indicate that C. thermocellum CbhA CBM4 also has an extended binding pocket that can optimally bind to cellodextrins containing five or more sugar units. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and, possibly, the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues Trp68 and Tyr110 play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose, and soluble oligodextrins. Binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that CBM4 binds the amorphous part or the cellulose “whiskers” of BMCC. We propose that clostridial CBM4s have possibly evolved to bind the free-chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins.     

Essential role of a family-32 carbohydrate-binding module in substrate recognition by Clostridium thermocellum mannanase CtMan5A

The family-5 glycoside hydrolase domain (GH5) and the family-32 carbohydrate-binding module (CBM32) of Clostridium thermocellum mannanase CtMan5A, along with their genetically inactivated derivatives, were collectively or separately expressed. Their catalytic and substrate-binding abilities were measured to investigate importance of CBM32 in substrate recognition by CtMan5A. Characterization of the truncated derivatives of CtMan5A and isothermal calorimetry analysis of the interaction between the inactivated proteins and mannooligosaccharides suggested that GH5 and CBM32 collectively formed a substrate-binding site capable of accommodating a mannotetraose unit in CtMan5A. This suggested that CBM32 directly participated in the substrate recognition required for catalytic action.     

Crystal structure of a cellulosomal family 3 carbohydrate esterase from Clostridium thermocellum provides insights into the mechanism of substrate recognition

The microbial degradation of the plant cell wall is of increasing industrial significance, exemplified by the interest in generating biofuels from plant cell walls. The majority of plant cell-wall polysaccharides are acetylated, and removal of the acetyl groups through the action of carbohydrate esterases greatly increases the efficiency of polysaccharide saccharification. Enzymes in carbohydrate esterase family 3 (CE3) are common in plant cell wall-degrading microorganisms but there is a paucity of structural and biochemical information on these biocatalysts. Clostridium thermocellum contains a single CE3 enzyme, CtCes3, which comprises two highly homologous (97% sequence identity) catalytic modules appended to a C-terminal type I dockerin that targets the esterase into the cellulosome, a large protein complex that catalyses plant cell wall degradation. Here, we report the crystal structure and biochemical properties of the N-terminal catalytic module (CtCes3-1) of CtCes3. The enzyme is a thermostable acetyl-specific esterase that exhibits a strong preference for acetylated xylan. CtCes3-1 displays an α/β hydrolase fold that contains a central five-stranded parallel twisted β-sheet flanked by six α-helices. In addition, the enzyme contains a canonical catalytic triad in which Ser44 is the nucleophile, His208 is the acid-base and Asp205 modulates the basic nature of the histidine. The acetate moiety is accommodated in a hydrophobic pocket and the negative charge of the tetrahedral transition state is stabilized through hydrogen bonds with the backbone N of Ser44 and Gly95 and the side-chain amide of Asn124.     

Structural insights of RmXyn10A – A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region

Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop β₆α₆ in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.     

Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase

The gene encoding the family 6 carbohydrate-binding module (CtCBM6A) from Clostridium thermocellum, cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant CtCBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of CtCBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (K _a of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (K _a of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, CtCBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant K _a of 230 M^?1 and binding capacity (N ₀) of 11 μmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58°C in the presence of Ca²⁺, indicating that Ca²⁺ imparts thermal stability to the CtCBM6A structure. Homology modeling of CtCBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of CtCBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that CtCBM6A has a stable conformation.     

Fine mapping and interaction analysis of a linear rabies virus neutralizing epitope

A novel human antibody AR16, targeting the G5 linear epitope of rabies virus glycoprotein (RVG) was shown to have promising antivirus potency. Using AR16, the minimal binding region within G5 was identified as HDFR (residues 261–264), with key residues HDF (residues 261–263) identified by alanine replacement scanning. The key HDF was highly conserved within phylogroup I Lyssaviruses but not those in phylogroup II. Using computer-aided docking and interaction models, not only the key residues (Asp30, Asp31, Tyr32, Trp53, Asn54, Glu99, Ile101, and Trp166) of AR16 that participated in the interaction with G5 were identified, the van der Waals forces that mediated the epitope–antibody interaction were also revealed. Seven out of eight presumed key residues (Asp30, Asp31, Tyr32, Trp53, Asn54, Glu99, and Ile101) were located at the variable regions of AR16 heavy chains. A novel mAb cocktail containing AR16 and CR57, has the potential to recognize non-overlapping, non-competing epitopes, and neutralize a broad range of rabies virus.     

Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module

The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE–CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE–CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.     

Mannan specific family 35 carbohydrate-binding module (CtCBM35) of Clostridium thermocellum: structure analysis and ligand binding

The three-dimensional model of the CtCBM35 (Cthe 2811), i.e. the family 35 carbohydrate binding module (CBM) from the Clostridium thermocellum family 26 glycoside hydrolase (GH) β-mannanase, generated by Modeller9v8 displayed predominance of β-sheets arranged as β-sandwich fold. Multiple sequence alignment of CtCBM35 with other CBM35s showed a conserved signature sequence motif Trp-Gly-Tyr, which is probably a specific determinant for mannan binding. Cloned CtCBM35 from Clostridium thermocellum ATCC 27405 was a homogenous, soluble 16 kDa protein. Ligand binding analysis of CtCBM35 by affinity electrophoresis displayed higher binding affinity against konjac glucomannan (K _a = 2.5 × 10⁵ M^?1) than carob galactomannan (K _a = 1.4 × 10⁵ M^?1). The presence of Ca²⁺ ions imparted slightly higher binding affinity of CtCBM35 against carob galactomannan and konjac glucomannan than without Ca²⁺ ion additive. However, CtCBM35 exhibited a low ligand-binding affinity K _a = 2.5 × 10^?5 M^?1 with insoluble ivory nut mannan. Ligand binding study by fluorescence spectroscopy showed K _a against konjac glucomannan and carob galactomannan, 2.4 × 10⁵ M^?1 and 1.44 × 10⁵ M^?1, and ΔG of binding ?27.0 and ?25.0 kJ/mol, respectively, substantiating the findings of affinity electrophoresis. Ca²⁺ ions escalated the thermostability of CtCBM35 and its melting temperature was shifted to 70°C from initial 55°C. Therefore thermostable CtCBM35 targets more β-(1,4)-manno-configured ligands from plant cell wall hemicellulosic reservoir. Thus a non-catalytic CtCBM35 of multienzyme cellulosomal enzymes may gain interest in the biofuel and food industry in the form of released sugars by targeting plant cell wall polysaccharides.     

Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities

Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as α, β and γ. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the γ sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of β and γ sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities.     

Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17

Crystal structures of chitosanase from Bacillus sp. K17 (ChoK) have been determined at 1.5 A resolution in the active form and at 2.0 A resolution in the inactive form. This enzyme belongs to the family GH-8, out of 93 glycoside hydrolase families, and exhibits the substrate specificity of subclass II chitosanase. The catalytic site is constructed on the scaffold of a double-alpha(6)/alpha(6)-barrel, which is formed by six repeating helix-loop-helix motifs. This structure is quite different from those of the GH-46 chitosanases and of GH-5. Structural comparison with CelA (a cellulase belonging to the same family GH-8) suggests that the proton donor Glu122 is conserved, but the proton acceptor is the inserted Glu309 residue, and that the corresponding Asp278 residue in CelA is inactivated in ChoK. The four acidic residues, Asp179, Glu309, Asp183 and Glu107, can be involved in substrate recognition through interactions with the amino groups of the glucosamine residues bound in the -3, -2, -1 and +1 sites, respectively. The hydrophobic Trp235, Trp166, Phe413 and Tyr318 residues are highly conserved for binding of the hexose rings at the -3, -2, +1 and +2 sites, respectively. These structural features indicate that enzymes in GH-8 can be further divided into three subfamilies. Different types of chitosanases are discussed in terms of convergent evolution from different structural ancestors.     

Crystal structure of hyperthermophilic endo-β-1,4-glucanase: implications for catalytic mechanism and thermostability

Endo-β-1,4-glucanase from thermophilic Fervidobacterium nodosum Rt17-B1 (FnCel5A), a new member of glycosyl hydrolase family 5, is highly thermostable and exhibits the highest activity on carboxymethylcellulose among the reported homologues. To understand the structural basis for the thermostability and catalytic mechanism, we report here the crystal structures of FnCel5A and the complex with glucose at atomic resolution. FnCel5A exhibited a (β/α)(8)-barrel structure typical of clan GH-A of the glycoside hydrolase families with a large and deep catalytic pocket located in the C-terminal end of the β-strands that may permit substrate access. A comparison of the structure of FnCel5A with related structures from thermopile Clostridium thermocellum, mesophile Clostridium cellulolyticum, and psychrophile Pseudoalteromonas haloplanktis showed significant differences in intramolecular interactions (salt bridges and hydrogen bonds) that may account for the difference in their thermostabilities. The substrate complex structure in combination with a mutagenesis analysis of the catalytic residues implicates a distinctive catalytic module Glu(167)-His(226)-Glu(283), which suggests that the histidine may function as an intermediate for the electron transfer network between the typical Glu-Glu catalytic module. Further investigation suggested that the aromatic residues Trp(61), Trp(204), Phe(231), and Trp(240) as well as polar residues Asn(51), His(127), Tyr(228), and His(235) in the active site not only participated in substrate binding but also provided a unique microenvironment suitable for catalysis. These results provide substantial insight into the unique characteristics of FnCel5A for catalysis and adaptation to extreme temperature.     

Mutational analysis of the carbohydrate binding activity of the tobacco lectin

At present the three-dimensional structure of the tobacco lectin, further referred to as Nictaba, and its carbohydrate-binding site are unresolved. In this paper, we propose a three-dimensional model for the Nictaba domain based on the homology between Nictaba and the carbohydrate-binding module 22 of Clostridium thermocellum Xyn10B. The suggested model nicely fits with results from circular dichroism experiments, indicating that Nictaba consists mainly of β-sheet. In addition, the previously identified nuclear localization signal is located at the top of the protein as a part of a protruding loop. Judging from this model and sequence alignments with closely related proteins, conserved glutamic acid and tryptophan residues in the Nictaba sequence were selected for mutational analysis. The mutant DNA sequences as well as the original Nictaba sequence have been expressed in Pichia pastoris and the recombinant proteins were purified from the culture medium. Subsequently, the recombinant proteins were characterized and their carbohydrate binding properties analyzed with glycan array technology. It was shown that mutation of glutamic acid residues in the C-terminal half of the protein did not alter the carbohydrate-binding activity of the lectin. In contrast, mutation of tryptophan residues in the N-terminal half of the Nictaba domain resulted in a complete loss of carbohydrate binding activity. These results suggest that tryptophan residues play an important role in the carbohydrate binding site of Nictaba.     

Binding characteristics of a bacterial expansin (BsEXLX1) for various types of pretreated lignocellulose

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus x giganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose.     

Theoretical calculations of the catalytic triad in short-chain alcohol dehydrogenases/reductases

Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD⁺ complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors.