The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes.

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)-linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M1 and anti-green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M1-mEGFP was expressed at levels equal to the M1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M1-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M1-mEGFP-expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M1-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

Selective phosphorylation of threonine residues defines GPR84-arrestin interactions of biased ligands.

GPR84 is an immune cell-expressed, proinflammatory receptor currently being assessed as a therapeutic target in conditions including fibrosis and inflammatory bowel disease. Although it was previously shown that the orthosteric GPR84 activators 2-HTP and 6-OAU promoted its interactions with arrestin-3, a G protein-biased agonist DL-175 did not. Here, we show that replacement of all 21 serine and threonine residues within i-loop 3 of GPR84, but not the two serines in the C-terminal tail, eliminated the incorporation of [32P] and greatly reduced receptor-arrestin-3 interactions promoted by 2-HTP. GPR84 was phosphorylated constitutively on residues Ser221 and Ser224, while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer221/pSer224 recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr263/pThr264 only recognized GPR84 after exposure to 2-HTP and not DL-175. Two distinct GPR84 antagonists as well as inhibition of G protein-coupled receptor kinase 2/3 prevented phosphorylation of pThr263/pThr264, but neither strategy affected constitutive phosphorylation of Ser221/Ser224. Furthermore, mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 also limited in 2-HTP-induced interactions with arrestin-2 and -3. By contrast, this mutant was unaffected in its capacity to reduce cAMP levels. Taken together, these results define a key pair of threonine residues, regulated only by subsets of GPR84 small molecule activators and by GRK2/3 that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84.

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t.

CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested that the receptor may exist as a dimer or an oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means, we confirm that CXCR4 can exist as a mixture of monomers, dimers, and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t-a small drug-like isothiourea derivative-rapidly destabilizes the oligomeric structure, whereas AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.

A general method to quantify ligand-driven oligomerization from fluorescence-based images.

Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein-protein interactions.

Design, Synthesis, and Evaluation of a Diazirine Photoaffinity Probe for Ligand-Based Receptor Capture Targeting G Protein-Coupled Receptors.

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein-coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography-mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P-NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P-binding receptor via liquid chromatography-tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors.

Nutrient and drought stress: implications for phenology and biomass quality in miscanthus.

BACKGROUND AND AIMS: The cultivation of dedicated biomass crops, including miscanthus, on marginal land provides a promising approach to the reduction of dependency on fossil fuels. However, little is known about the impact of environmental stresses often experienced on lower-grade agricultural land on cell-wall quality traits in miscanthus biomass crops. In this study, three different miscanthus genotypes were exposed to drought stress and nutrient stress, both separately and in combination, with the aim of evaluating their impact on plant growth and cell-wall properties. METHODS: Automated imaging facilities at the National Plant Phenomics Centre (NPPC-Aberystwyth) were used for dynamic phenotyping to identify plant responses to separate and combinatorial stresses. Harvested leaf and stem samples of the three miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus and Miscanthus × giganteus) were separately subjected to saccharification assays, to measure sugar release, and cell-wall composition analyses. KEY RESULTS: Phenotyping showed that the M. sacchariflorus genotype Sac-5 and particularly the M. sinensis genotype Sin-11 coped better than the M. × giganteus genotype Gig-311 with drought stress when grown in nutrient-poor compost. Sugar release by enzymatic hydrolysis, used as a biomass quality measure, was significantly affected by the different environmental conditions in a stress-, genotype- and organ-dependent manner. A combination of abundant water and low nutrients resulted in the highest sugar release from leaves, while for stems this was generally associated with the combination of drought and nutrient-rich conditions. Cell-wall composition analyses suggest that changes in fine structure of cell-wall polysaccharides, including heteroxylans and pectins, possibly in association with lignin, contribute to the observed differences in cell-wall biomass sugar release. CONCLUSIONS: The results highlight the importance of the assessment of miscanthus biomass quality measures in addition to biomass yield determinations and the requirement for selecting suitable miscanthus genotypes for different environmental conditions.

Functionalization of paramagnetic nanoparticles for protein immobilization and purification.

A paramagnetic nanocomposite coated with chitosan and N-(5-Amino-1-carboxy-pentyl) iminodiacetic acid (NTA) that is suitable for protein immobilization applications has been prepared and characterized. The nanoparticle core was synthesized by controlled aggregation of Fe3O4 under alkaline conditions, and Transmission Electron Microscopy revealed a size distribution of 10-50 nm. The nanoparticle core was coated with chitosan and derivatized with glutaraldehyde and NTA, as confirmed by Fourier Transform Infrared Spectroscopy. The final nanoparticles were used as a metal affinity matrix to separate a recombinant polyhistidine-tagged ß-galactosidase from Bacillus subtilis directly from E. coli cell lysates with high purity (>95%). After loading with Ni2+, nanoparticles demonstrated a binding capacity of 250 µg of a polyhistidine-tagged ß-galactosidase per milligram of support. The immobilized enzyme retained 80% activity after 9 cycles of washing, and the immobilized recombinant protein could be eluted with high purity with imidazole. The applications for these nanomagnetic composites extend beyond protein purification, and can also be used for immobilizing enzymes, where the ß-galactosidase immobilized on the nanomagnetic support was used in multiple cycles of catalytic reactions with no significant loss of catalytic activity.

Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor.

Previous studies have indicated that the G-protein-coupled secretin receptor is present as a homodimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high-potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and spatial intensity distribution analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed us to demonstrate that the epidermal growth factor receptor is predominantly monomeric in the absence of ligand and while wild-type receptor was rapidly converted into a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that, at moderate expression levels, the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate-induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. In contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G-protein Gαs did not alter receptor organization, indicating that dimerization is defined specifically by direct protein-protein interactions between copies of the receptor polypeptide, while short-term treatment with secretin had no effect on organization of the wild-type receptor but increased the dimeric proportion of the mutated receptor variant.

Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs.

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·µm(-2) human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior.

Regulation of oligomeric organization of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor observed by spatial intensity distribution analysis.

The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor.

The molecular basis of oligomeric organization of the human M3 muscarinic acetylcholine receptor.

G protein-coupled receptors, including the M3 muscarinic acetylcholine receptor, can form homo-oligomers. However, the basis of these interactions and the overall organizational structure of such oligomers are poorly understood. Combinations of site-directed mutagenesis and homogenous time-resolved fluorescence resonance energy transfer studies that assessed interactions between receptor protomers at the surface of transfected cells indicated important contributions of regions of transmembrane domains I, IV, V, VI, and VII as well as intracellular helix VIII to the overall organization. Molecular modeling studies based on both these results and an X-ray structure of the inactive state of the M3 receptor bound by the antagonist/inverse agonist tiotropium were then employed. The results could be accommodated fully by models in which a proportion of the cell surface M3 receptor population is a tetramer with rhombic, but not linear, orientation. This is consistent with previous studies based on spectrally resolved, multiphoton fluorescence resonance energy transfer. Modeling studies furthermore suggest an important role for molecules of cholesterol at the dimer + dimer interface of the tetramer, which is consistent with the presence of cholesterol at key locations in many G protein-coupled receptor crystal structures. Mutants that displayed disrupted quaternary organization were often poorly expressed and showed immature N-glycosylation. Sustained treatment of cells expressing such mutants with the muscarinic receptor inverse agonist atropine increased cellular levels and restored both cell surface delivery and quaternary organization to many of the mutants. These observations suggest that organization as a tetramer may occur before plasma membrane delivery and may be a key step in cellular quality control assessment.

Structural and biophysical characterisation of G protein-coupled receptor ligand binding using resonance energy transfer and fluorescent labelling techniques.

The interaction between ligands and the G protein-coupled receptors (GPCRs) to which they bind has long been the focus of intensive investigation. The signalling cascades triggered by receptor activation, due in most cases to ligand binding, are of great physiological and medical importance; indeed, GPCRs are targeted by in excess of 30% of small molecule therapeutic medicines. Attempts to identify further pharmacologically useful GPCR ligands, for receptors with known and unknown endogenous ligands, continue apace. In earlier days direct assessment of such interactions was restricted largely to the use of ligands incorporating radioactive isotope labels as this allowed detection of the ligand and monitoring its interaction with the GPCR. This use of such markers has continued with the development of ligands labelled with fluorophores and their application to the study of receptor-ligand interactions using both light microscopy and resonance energy transfer techniques, including homogenous time-resolved fluorescence resonance energy transfer. Details of ligand-receptor interactions via X-ray crystallography are advancing rapidly as methods suitable for routine production of substantial amounts and stabilised forms of GPCRs have been developed and there is hope that this may become as routine as the co-crystallisation of serine/threonine kinases with ligands, an approach that has facilitated widespread use of rapid structure-based ligand design. Conformational changes involved in the activation of GPCRs, widely predicted by biochemical and biophysical means, have inspired the development of intramolecular FRET-based sensor forms of GPCRs designed to investigate the events following ligand binding and resulting in a signal propagation across the cell membrane. Finally, a number of techniques are emerging in which ligand-GPCR binding can be studied in ways that, whilst indirect, are able to monitor its results in an unbiased and integrated manner. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.

Enhanced xyloglucan-specific endo-ß-1,4-glucanase efficiency in an engineered CBM44-XegA chimera.

Xyloglucan-specific endo-ß-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for ß-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.

Roundabout 1 exists predominantly as a basal dimeric complex and this is unaffected by binding of the ligand Slit2.

Robo (Roundabout) receptors and their Slit polypeptide ligands are known to play key roles in neuronal development and have been implicated in both angiogenesis and cancer. Like the other family members, Robo1 is a large single transmembrane domain polypeptide containing a series of well-defined extracellular elements. However, the intracellular domain lacks structural definition and little is known about the quaternary structure of Robo receptors or how binding of a Slit might affect this. To address these questions combinations of both autofluorescent protein-based FRET imaging and time-resolved FRET were employed. Both approaches identified oligomeric organization of Robo1 that did not require the presence of the intracellular domain. SpIDA (spatial intensity distribution analysis) of eGFP-tagged forms of Robo1 indicated that for a C-terminally deleted version approximately two-thirds of the receptor was present as a dimer and one-third as a monomer. By contrast, full-length Robo1 was present almost exclusively as a dimer. In each case this was unaffected by the addition of Slit2, although parallel studies demonstrated the biological activity of Slit2 and its interaction with Robo1. Deletion of both the immunoglobulin and fibronectin type III extracellular repeats prevented dimer formation, with the immunoglobulin repeats providing the bulk of the protein-protein interaction affinity.

Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor.

Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells─The Impact of Ligands and Membrane Disruptors.

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different ligand states, together with spectroscopic studies and molecular dynamics simulations, have revealed a rich conformational landscape of GPCRs. However, their supramolecular structure and spatiotemporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching, and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M1 receptor (M1R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M1R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M1 receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile (∼0.01 µm2/s), slow (∼0.04 µm2/s), and fast (∼0.14 µm2/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6 ceramide led to significant changes for CD86, while the diffusion of M1R remained unchanged, indicating that M1 receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression and the binding of orthosteric ligands; in particular, the agonist carbachol elicited a large increase in the fraction of M1R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.

Intramolecular fluorescence resonance energy transfer (FRET) sensors of the orexin OX1 and OX2 receptors identify slow kinetics of agonist activation.

Intramolecular fluorescence resonance energy transfer (FRET) sensors able to detect changes in distance or orientation between the 3rd intracellular loop and C-terminal tail of the human orexin OX(1) and OX(2) G protein-coupled receptors following binding of agonist ligands were produced and expressed stably. These were directed to the plasma membrane and, despite the substantial sequence alterations introduced, in each case were able to elevate [Ca(2+)](i), promote phosphorylation of the ERK1/2 MAP kinases and become internalized effectively upon addition of the native orexin peptides. Detailed characterization of the OX(1) sensor demonstrated that it was activated with rank order of potency orexin A > orexin B > orexin A 16-33, that it bound antagonist ligands with affinity similar to the wild-type receptor, and that mutation of a single residue, D203A, greatly reduced the binding and function of orexin A but not antagonist ligands. Addition of orexin A to individual cells expressing an OX(1) sensor resulted in a time- and concentration-dependent reduction in FRET signal consistent with mass-action and potency/affinity estimates for the peptide. Compared with the response kinetics of a muscarinic M(3) acetylcholine receptor sensor upon addition of agonist, response of the OX(1) and OX(2) sensors to orexin A was slow, consistent with a multistep binding and activation process. Such sensors provide means to assess the kinetics of receptor activation and how this may be altered by mutation and sequence variation of the receptors.

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-ß-1,4-glucanase (GH12) from Aspergillus niveus.

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-ß-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan ß-1,3 or ß-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in ß-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168.

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

The feruloyl esterase from Thermobacillus xylanilyticus shows broad specificity for processing pre-biotic feruloylated xylooligosaccharides at high temperatures.

Ferulic acid has antioxidant properties of interest to the food industry and can be released from natural plant fibres using feruloyl esterases. Esterases active at high temperatures are highly desirable but currently underrepresented. Here we report the biochemical characterization of the feruloyl esterase from Thermobacillus xylanilyticus. Specific activity of recombinant Tx-Est1 with ethyl ferulate was 29.2 ± 2.9 U mg-1, with a catalytic efficiency (Kcat/Km) of 393.7 ± 9.8 s-1mM-1. The temperature and pH optima were 60 °C and 7.5, whereby Tx-Est1 retains 70% activity after 25 h at 40 °C. MALDI-TOF MS revealed Tx-ESTI released ferulic acid from xylooligosaccharides with DP4-DP13, and from DP6-8 containing two ferulic acid groups. HPLC demonstrated ferulic acid release from destarched wheat bran was strongly potentiated by co-incubation with xylanase. These properties, especially the high activity at elevated temperatures, suggest Tx-Est1 can be employed for production of high-value compounds from agricultural waste or during plant polysaccharide saccharification.