Binding mechanisms for Thermobifida fusca Cel5A,Cel6B,and Cel48A cellulose-binding modules on bacterial microcrystalline cellulose

The family II cellulose-binding modules (CBM) from Thermobifida fusca Cel5A and Cel48A were cloned in the Escherichia coli/Streptomyces shuttle vector pD730, and the plasmids were transformed into Streptomyces lividans TKM31. CBM(Cel5A), and CBM(Cel48A), CBM(Cel6B) were expressed and purified from S. lividans. The molecular masses were determined by mass spectrometry, and the values were 10595 +/- 2, 10915 +/- 2, and 11291 +/- 2 Da for CBM(Cel5A), CBM(Cel6B), and CBM(Cel48A), respectively. Three different binding models (Langmuir, Interstice Penetration, and Interstice Saturation) were tested to describe the binding isotherms of these CBMs on bacterial microcrystalline cellulose (BMCC). The experimental binding isotherms of T. fusca family II CBMs on BMCC are best modeled by the Interstice Saturation model, which includes binding to the constrained interstice surface of BMCC as well as traditional Langmuir binding on the freely accessible surface. The Interstice Saturation model consists of three different steps (Langmuir binding, interstice binding, and interstice saturation). Full reversibility only occurred in the Langmuir region. The irreversibility in the interstice binding and saturation regions probably was caused by interstice entrapment. Temperature shift experiments in different binding regions support the interstice entrapment assumption. There was no systematic difference in binding between the two types of exocellulase CBMs--one that hydrolyzes cellulose from the nonreducing (CBM(Cel6B)) end and one that hydrolyzes cellulose from the reducing end (CBM(Cel48A)).     

Binding and reversibility of Thermobifida fusca Cel5A,Cel6B,and Cel48A and their respective catalytic domains to bacterial microcrystalline cellulose

The binding and reversibility of Thermobifida fusca intact Cel5A, Cel5B, and Cel48A and their corresponding catalytic domains (CDs) to bacterial microcrystalline cellulose (BMCC) were studied at 5 degrees C. The binding of the intact cellulases and of corresponding CDs to BMCC was irreversible in all regions: Langmuir binding (region I), interstice penetration (region II), and interstice saturation (region III). The three cellulose binding domains (CBMs) bind reversibly in "region I" although their respective CDs do not. The irreversible binding of these enzymes in the Langmuir region does not satisfy the Langmuir assumption; however, the overall fit of the Interstice Saturation model, which includes binding in MBCC interstices as well as on the freely accessible surface (Jung et al., 2002a) is good. The main limitation of the model is that it does not explicitly address a mechanism for forming the enzyme-substrate complex within the active site of the CDs.     

Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.

Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.     

Hypocrea jecorina (Trichoderma reesei) Cel7A as a molecular machine: A docking study

Hypocrea jecorina (formerly Trichoderma reesei) Cel7A has a catalytic domain (CD) and a cellulose-binding domain (CBD) separated by a highly glycosylated linker. Very little is known of how the 2 domains interact to degrade crystalline cellulose. Based on the interaction energies and forces on cello-oligosaccharides computationally docked to the CD and CBD, we propose a molecular machine model, where the CBD wedges itself under a free chain end on the crystalline cellulose surface and feeds it to the CD active site tunnel. Enzyme-substrate interactions produce the forces required to pull cellulose chains from the surface and also to help the enzyme move on the cellulose chain for processive hydrolysis. The energy to generate these forces is ultimately derived from the chemical energy of glycosidic bond breakage.     

Cell wall targeted in planta iron accumulation enhances biomass conversion and seed iron concentration in Arabidopsis and rice

Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion‐limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM‐IBP fusion polypeptides composed of a carbohydrate‐binding module family 11 (CBM11) and an iron‐binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM‐IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM‐IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM‐IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.     

Starch-synthase III family encodes a tandem of three starch-binding domains

The starch-synthase III (SSIII), with a total of 1025 residues, is one of the enzymes involved in plants starch synthesis. SSIII from Arabidopsis thaliana contains a putative N-terminal transit peptide followed by a 557-amino acid SSIII-specific domain (SSIII-SD) with three internal repeats and a C-terminal catalytic domain of 450 amino acids. Here, using computational characterization techniques, we show that each of the three internal repeats encodes a starch-binding domain (SBD). Although the SSIII from A. thaliana and its close homologous proteins show no detectable sequence similarity with characterized SBD sequences, the amino acid residues known to be involved in starch binding are well conserved.     

Structural basis for cellulose binding by the type A carbohydrate‐binding module 64 of Spirochaeta thermophila

Spirochaeta thermophila secretes seven glycoside hydrolases for plant biomass degradation that carry a carbohydrate‐binding module 64 (CBM64) appended at the C‐terminus. CBM64 adsorbs to various β1‐4‐linked pyranose substrates and shows high affinity for cellulose. We present the first crystal structure of a CBM64 at 1.2 Å resolution, which reveals a jelly‐roll‐like fold corresponding to a surface‐binding type A CBM. Modeling of its interaction with cellulose indicates that CBM64 achieves association with the hydrophobic face of β‐linked pyranose chains via a unique coplanar arrangement of four exposed tryptophan side chains. Proteins 2016; 84:855–858. © 2016 Wiley Periodicals, Inc.     

Dimensions and specificities of recognition sites on lectins and antibodies

A comparison is made of the specific combining sites of a number of lectins and of antibodies with emphasis on those reacting with blood group A, B, and H determinants. The ranges of site sizes and specificities of both groups are similar both from immunochemical studies and from the limited x-ray diffraction data available.     

Crystallographic, thermodynamic, and molecular modeling studies of the mode of binding of oligosaccharides to the potent antiviral protein griffithsin

The mode of binding of oligosaccharides to griffithsin, an antiviral lectin from the red alga Griffithsia sp., was investigated by a combination of X-ray crystallography, isothermal titration calorimetry, and molecular modeling. The structures of complexes of griffithsin with 1-->6alpha-mannobiose and with maltose were solved and refined at the resolution of 2.0 and 1.5 A, respectively. The thermodynamic parameters of binding of 1-->6alpha-mannobiose, maltose, and mannose to griffithsin were determined. Binding profiles of 1-->6alpha-mannobiose and mannose were similar with Kd values of 83.3 microM and 102 microM, respectively. The binding of maltose to griffithsin was significantly weaker, with a fourfold lower affinity (Kd = 394 microM). In all cases the binding at 30 degrees C was entropically rather than enthalpically driven. On the basis of the experimental crystal structures, as well as on previously determined structures of complexes with monosaccharides, it was possible to create a model of a tridentate complex of griffithsin with Man9GlcNAc2, a high mannose oligosaccharide commonly found on the surface of viral glycoproteins. All shorter oligomannoses could be modeled only as bidentate or monodentate complexes with griffithsin. The ability to mediate tight multivalent and multisite interactions with high-mannose oligosaccharides helps to explain the potent antiviral activity of griffithsin.     

Cooperative and competitive binding in synergistic mixtures of Thermobifida fusca cellulases Cel5A,Cel6B,and Cel9A

Synergism between cellulases facilitates efficient hydrolysis of microcrystalline cellulose. We hypothesize that the effects of synergism, observed as enhanced extents of hydrolysis, are related to cellulase binding to the substrate in mixtures. In this study, direct measurements of bound concentrations of fluorescence-labeled T. fusca Cel5A, Cel6B, and Cel9A on bacterial microcrystalline cellulose were used to study binding behaviors of cellulases in binary component reactions. The accuracy of the determination of fluorescence-labeled cellulase concentrations in binary component mixtures was in the range of 7-9%. Data at 5 degrees C show that binding levels of cellulases in mixture reactions are only 22-70% of the binding levels in single component reactions. At 50 degrees C, however, most of the cellulase components in the same mixtures bound to extents of 40-126% higher than in the corresponding single component reactions. The degrees of synergistic effect (DSE) observed for the reactions at 50 degrees C were greater than 1, indicating that the components in the mixture acted synergistically, whereas DSE < 1 was generally observed for the reactions at 5 degrees C indicating anti-synergistic behavior. Degrees of synergistic binding (DSB) were also calculated, where anti-synergistic mixtures had DSB < 1 and synergistic mixtures had DSB>1. We conclude that the lower extents of binding at 5 degrees C are due to competition for binding sites by the cellulase components in the mixtures and the enhanced binding extents at 50 degrees C are due to increased availability of binding sites on the substrates brought about by the higher extents of hydrolysis.     

Interaction between the CBM of Cel9A from Thermobifida fusca and cellulose fibers

Molecular docking and molecular dynamics (MD) simulations were used to investigate the binding of a cellodextrin chain in a crystal-like conformation to the carbohydrate-binding module (CBM) of Cel9A from Thermobifida fusca. The fiber was found to bind to the CBM in a single and well-defined configuration in-line with the catalytic cleft, supporting the hypothesis that this CBM plays a role in the catalysis by feeding the catalytic domain (CD) with a polysaccharide chain. The results also expand the current known list of residues involved in the binding. The polysaccharide-protein attachment is shown to be mediated by five amine/amide-containing residues. E478 and E559 were found not to interact directly with the sugar chain; instead they seem to be responsible to stabilize the binding motif via hydrogen bonds.     

Computational models explain the oligosaccharide specificity of cyanovirin-N

The prokaryotic lectin cyanovirin-N (CV-N) is a potent inhibitor of HIV envelope-mediated cell entry, and thus is a leading candidate among a new class of potential anti-HIV microbicides. The activity of CV-N is a result of interactions with the D1 arm of high-mannose oligosaccharides on the viral glycoprotein gp120. Here, we present computationally refined models of CV-N recognition of the di- and trisaccharides that represent the terminal three sugars of the D1 arm by each CV-N binding site. These models complement existing structural data, both from NMR spectroscopy and X-ray crystallography. When used with a molecular dynamics/continuum electrostatic (MD/PBSA) approach to compute binding free energies, these models explain the relative affinity of each site for the two saccharides. This work presents the first validation of the application of continuum electrostatic models to carbohydrate-protein association. Taken as a whole, the results both provide models of CV-N sugar recognition and demonstrate the utility of these computational methods for the study of carbohydrate-binding proteins.     

Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures

Thermostability is an important feature in industrial enzymes: it increases biocatalyst lifetime and enables reactions at higher temperatures, where faster rates and other advantages ultimately reduce the cost of biocatalysis. Here we report the thermostabilization of a chimeric fungal family 6 cellobiohydrolase (HJPlus) by directed evolution using random mutagenesis and recombination of beneficial mutations. Thermostable variant 3C6P has a half‐life of 280 min at 75°C and a T₅₀ of 80.1°C, a ～15°C increase over the thermostable Cel6A from Humicola insolens (HiCel6A) and a ～20°C increase over that from Hypocrea jecorina (HjCel6A). Most of the mutations also stabilize the less‐stable HjCel6A, the wild‐type Cel6A closest in sequence to 3C6P. During a 60‐h Avicel hydrolysis, 3C6P released 2.4 times more cellobiose equivalents at its optimum temperature (T_opt) of 75°C than HiCel6A at its T_opt of 60°C. The total cellobiose equivalents released by HiCel6A at 60°C after 60 h is equivalent to the total released by 3C6P at 75°C after ～6 h, a 10‐fold reduction in hydrolysis time. A binary mixture of thermostable Cel6A and Cel7A hydrolyzes Avicel synergistically and released 1.8 times more cellobiose equivalents than the wild‐type mixture, both mixtures assessed at their respective T_opt. Crystal structures of HJPlus and 3C6P, determined at 1.5 and 1.2 Å resolution, indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by introduced proline residues. Biotechnol. Bioeng. 2013; 110: 1874–1883. © 2013 Wiley Periodicals, Inc.     

Characteristics of the binding of a bacterial expansin (BsEXLX1) to microcrystalline cellulose

Plant expansin proteins induce plant cell wall extension and have the ability to extend and disrupt cellulose. In addition, these proteins show synergistic activity with cellulases during cellulose hydrolysis. BsEXLX1 originating from Bacillus subtilis is a structural homolog of a β‐expansin produced by Zea mays (ZmEXPB1). The Langmuir isotherm for binding of BsEXLX1 to microcrystalline cellulose (i.e., Avicel) revealed that the equilibrium binding constant of BsEXLX1 to Avicel was similar to those of other Type A surface‐binding carbohydrate‐binding modules (CBMs) to microcrystalline cellulose, and the maximum number of binding sites on Avicel for BsEXLX1 was also comparable to those on microcrystalline cellulose for other Type A CBMs. BsEXLX1 did not bind to cellooligosaccharides, which is consistent with the typical binding behavior of Type A CBMs. The preferential binding pattern of a plant expansin, ZmEXPB1, to xylan, compared to cellulose was not exhibited by BsEXLX1. In addition, the binding capacities of cellulose and xylan for BsEXLX1 were much lower than those for CtCBD3. Biotechnol. Bioeng. 2013; 110: 401–407. © 2012 Wiley Periodicals, Inc.     

Functional and structural studies of pullulanase from Anoxybacillus sp. LM18‐11

Pullulanase is a debranching enzyme that specifically hydrolyzes the α‐1,6 glycosidic linkage of α‐glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18‐11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and K_m for pullulan of PulA are 750 U mg^?1 and 16.4 μmol L^?1, respectively. PulA has a half‐life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75–2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β‐strand and the third α‐helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate‐binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685–1693. © 2013 Wiley Periodicals, Inc.     

A Bitter Principle of Asparagus: Isolation and Structure of Furostanol Saponin in Asparagus Storage Root

During an examination of components contributing to the bitter taste of asparagus bottom cut (Asparagus officinalis L.), two new furostanol saponins were isolated from roots extractives. Their chemical structures were established as 5β-furostane-3β,22,26 triol-3-O-β-d-glucopyranosyl (1→2)-β-d-glucopyranoside 26-O-β-d-glucopyranoside and 5β-furostane-3β,22,26 triol-3-O-β-d-glucopyranosyl (1→2) [β-d-xylopyranoxyl (1→4)]-β-d-glucopyranoside 26-O-β-d-glucopyranoside respectively.     

Structure-function analysis of endogenous lectin mind-the-gap in synaptogenesis

Mind-the-Gap (MTG) is required for neuronal induction of Drosophila neuromuscular junction (NMJ) postsynaptic domains, including glutamate receptor (GluR) localization. We have previously hypothesized that MTG is secreted from the presynaptic terminal to reside in the synaptic cleft, where it binds glycans to organize the heavily glycosylated, extracellular synaptomatrix required for transsynaptic signaling between neuron and muscle. In this study, we test this hypothesis with MTG structure-function analyses of predicted signal peptide (SP) and carbohydrate-binding domain (CBD), by introducing deletion and point-mutant transgenic constructs into mtg null mutants. We show that the SP is required for MTG secretion and localization to synapses in vivo. We further show that the CBD is required to restrict MTG diffusion in the extracellular synaptomatrix and for postembryonic viability. However, CBD mutation results in elevation of postsynaptic GluR localization during synaptogenesis, not the mtg null mutant phenotype of reduced GluRs as predicted by our hypothesis, suggesting that proper synaptic localization of MTG limits GluR recruitment. In further testing CBD requirements, we show that MTG binds N-acetylglucosamine (GlcNAc) in a Ca(2+)-dependent manner, and thereby binds HRP-epitope glycans, but that these carbohydrate interactions do not require the CBD. We conclude that the MTG lectin has both positive and negative binding interactions with glycans in the extracellular synaptic domain, which both facilitate and limit GluR localization during NMJ embryonic synaptogenesis.     

A Comparison of Soybean Globulins and the Protein Bodies in the Protein Composition

The protein compositions between soybean globulins and the protein bodies were compared by gel filtration with Sephadex G-200, sedimentation analyses and disc isoelectric focusing.

From the results of the three comparisons, it was difficult to find an essential difference in the protein compositions of the both. And, the 7S and the 11S globulins were the main components in the both. This fact was more strongly suggested that these globulins were the typical reserve proteins.