Hamiltonian Dynamics and Structural States of Two-Dimensional Active Particles.

We show that a two-dimensional system of flocking active particles interacting hydrodynamically can be expressed using a Hamiltonian formalism. The Hamiltonian depends strictly on the angles between the particles and their orientation, thereby restricting their available phase-space. Simulations of co-oriented active particles evolve into "escalators"-sharp lines at a particular tilt along which particles circulate. The conservation of the Hamiltonian and its symmetry germinate the self-assembly of the observed steady-state arrangements as confirmed by stability analysis.

Deciphering Cellodextrin and Glucose Uptake in Clostridium thermocellum.

Sugar uptake is of great significance in industrially relevant microorganisms. Clostridium thermocellum has extensive potential in lignocellulose biorefineries as an environmentally prominent, thermophilic, cellulolytic bacterium. The bacterium employs five putative ATP-binding cassette transporters which purportedly take up cellulose hydrolysates. Here, we first applied combined genetic manipulations and biophysical titration experiments to decipher the key glucose and cellodextrin transporters. In vivo gene inactivation of each transporter and in vitro calorimetric and nuclear magnetic resonance (NMR) titration of each putative sugar-binding protein with various saccharides supported the conclusion that only transporters A and B play the roles of glucose and cellodextrin transport, respectively. To gain insight into the structural mechanism of the transporter specificities, 11 crystal structures, both alone and in complex with appropriate saccharides, were solved for all 5 putative sugar-binding proteins, thus providing detailed specific interactions between the proteins and the corresponding saccharides. Considering the importance of transporter B as the major cellodextrin transporter, we further identified its cryptic, hitherto unknown ATPase-encoding gene as clo1313_2554, which is located outside the transporter B gene cluster. The crystal structure of the ATPase was solved, showing that it represents a typical nucleotide-binding domain of the ATP-binding cassette (ABC) transporter. Moreover, we determined that the inducing effect of cellobiose (G2) and cellulose on cellulosome production could be eliminated by deletion of transporter B genes, suggesting the coupling of sugar transport and regulation of cellulosome components. This study provides key basic information on the sugar uptake mechanism of C. thermocellum and will promote rational engineering of the bacterium for industrial application. IMPORTANCE Highly efficient sugar uptake is important to microbial cell factories, and sugar transporters are therefore of great interest in the study of industrially relevant microorganisms. Clostridium thermocellum is a lignocellulolytic bacterium known for its multienzyme complex, the cellulosome, which is of great potential value in lignocellulose biorefinery. In this study, we clarify the function and mechanism of substrate specificity of the five reported putative sugar transporters using genetic, biophysical, and structural methods. Intriguingly, the results showed that only one of them, transporter B, is the major cellodextrin transporter, whereas another, transporter A, represents the major glucose transporter. Considering the importance of transporter B, we further identified the missing ATPase gene of transporter B and revealed the correlation between transporter B and cellulosome production. Revealing the mechanism by which C. thermocellum utilizes cellodextrins will help pave the way for engineering the strain for industrial applications.

Integrative structure determination reveals functional global flexibility for an ultra-multimodular arabinanase.

AbnA is an extracellular GH43 α-L-arabinanase from Geobacillus stearothermophilus, a key bacterial enzyme in the degradation and utilization of arabinan. We present herein its full-length crystal structure, revealing the only ultra-multimodular architecture and the largest structure to be reported so far within the GH43 family. Additionally, the structure of AbnA appears to contain two domains belonging to new uncharacterized carbohydrate-binding module (CBM) families. Three crystallographic conformational states are determined for AbnA, and this conformational flexibility is thoroughly investigated further using the "integrative structure determination" approach, integrating molecular dynamics, metadynamics, normal mode analysis, small angle X-ray scattering, dynamic light scattering, cross-linking, and kinetic experiments to reveal large functional conformational changes for AbnA, involving up to ~100 Å movement in the relative positions of its domains. The integrative structure determination approach demonstrated here may apply also to the conformational study of other ultra-multimodular proteins of diverse functions and structures.

Cross-utilization of ß-galactosides and cellobiose in Geobacillus stearothermophilus.

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-ß-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-ß-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

Carbohydrate-Binding Capability and Functional Conformational Changes of AbnE, an Arabino-oligosaccharide Binding Protein.

ABC importers are membrane proteins responsible for the transport of nutrients into the cells of prokaryotes. Although the structures of ABC importers vary, all contain four conserved domains: two nucleotide-binding domains (NBDs), which bind and hydrolyze ATP, and two transmembrane domains (TMDs), which help translocate the substrate. ABC importers are also dependent on an additional protein component, a high-affinity substrate-binding protein (SBP) that specifically binds the target ligand for delivery to the appropriate ABC transporter. AbnE is a SBP belonging to the ABC importer for arabino-oligosaccharides in the Gram-positive thermophilic bacterium Geobacillus stearothermophilus. Using isothermal titration calorimetry (ITC), purified AbnE was shown to bind medium-sized arabino-oligosaccharides, in the range of arabino-triose (A3) to arabino-octaose (A8), all with Kd values in the nanomolar range. We describe herein the 3D structure of AbnE in its closed conformation in complex with a wide range of arabino-oligosaccharide substrates (A2-A8). These structures provide the basis for the detailed structural analysis of the AbnE-sugar complexes, and together with complementary quantum chemical calculations, site-specific mutagenesis, and isothermal titration calorimetry (ITC) experiments, provide detailed insights into the AbnE-substrate interactions involved. Small-angle X-ray scattering (SAXS) experiments and normal mode analysis (NMA) are used to study the conformational changes of AbnE, and these data, taken together, suggest clues regarding its binding mode to the full ABC importer.

Spectroscopic FTIR and NMR study of the interactions of sugars with proteins.

FTIR and NMR spectra were measured in parallel for specific two-components mixtures of various proteins with different sugar molecules, such as arabinose, glucose, and sucrose. In the FTIR spectra of arabinose with some of these proteins, the bands assigned to the vibrational modes of the CH and COH groups disappeared, and new ones, related to an arabinose-protein CN mode, appeared. Similar changes were observed in the FTIR spectra of lyophilized mixtures of arabinose with different amino acids. In additional FTIR spectra, measured for other protein-sugar mixtures, the bands correlated to the ring modes of arabinose, in the range 1150-1000 cm-1, disappeared, and two new very strong narrow bands became dominant, indicating ring opening or some kind of arabinose decomposition. Contrary to the prevailing opinion that complexes between sugars and proteins are formed mainly by hydrogen bonds, the IR and NMR spectra of the sugar-protein mixtures studied here suggest that significant chemical reactions also take place between the interacting sugar and the protein. Two types of sugar-protein chemical reactions can be distinguished on the basis of these IR spectra, leading to the formation of a new CN bond and to the decomposition of sugar skeletal bonds. The new IR bands suggest that the latter reaction results in the formation of new bonds, which are related to new polyether moieties. These results highlight the often ignored non-specific chemical reactions that take place between sugars and proteins, and demonstrate that the simultaneous application of FTIR and NMR spectroscopic analyses can detect and further characterize these types of sugar-protein interactions.

Regulation of biomass degradation by alternative σ factors in cellulolytic clostridia.

Bacteria can adjust their genetic programs via alternative σ factors to face new environmental pressures. Here, we analyzed a unique set of paralogous alternative σ factors, termed σIs, which fine-tune the regulation of one of the most intricate cellulolytic systems in nature, the bacterial cellulosome, that is involved in degradation of environmental polysaccharides. We combined bioinformatics with experiments to decipher the regulatory networks of five σIs in Clostridium thermocellum, the epitome of cellulolytic microorganisms, and one σI in Pseudobacteroides cellulosolvens which produces the cellulosomal system with the greatest known complexity. Despite high homology between different σIs, our data suggest limited cross-talk among them. Remarkably, the major cross-talk occurs within the main cellulosomal genes which harbor the same σI-dependent promoter elements, suggesting a promoter-based mechanism to guarantee the expression of relevant genes. Our findings provide insights into the mechanisms used by σIs to differentiate among their corresponding regulons, representing a comprehensive overview of the regulation of the cellulosome to date. Finally, we show the advantage of using a heterologous host system for analysis of multiple σIs, since information generated by their analysis in their natural host can be misinterpreted owing to a cascade of interactions among the different σIs.

Assembly of Synthetic Functional Cellulosomal Structures onto the Cell Surface of Lactobacillus plantarum, a Potent Member of the Gut Microbiome.

Heterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the "adaptor scaffoldin" strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia of Lactobacillus plantarum, a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all produced in vivo by the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw.IMPORTANCE The multiple benefits of lactic acid bacteria are well established in health and industry. Here we present an approach designed to extensively increase the cell surface display of proteins via successive assembly of interactive components. Our findings present a stepping stone toward proficient engineering of Lactobacillus plantarum, a widespread, environmentally important bacterium and potent microbiome member, for improved degradation of lignocellulosic biomass and advanced probiotics.

Structural basis for enzyme bifunctionality - the case of Gan1D from Geobacillus stearothermophilus.

6-phospho-ß-glucosidases and 6-phospho-ß-galactosidases are enzymes that hydrolyze the ß-glycosidic bond between a terminal non-reducing glucose-6-phosphate (Glc6P) or galactose-6-phosphate (Gal6P), respectively, and other organic molecules. Gan1D, a glycoside hydrolase (GH) belonging to the GH1 family, has recently been identified in a newly characterized galactan-utilization gene cluster in the bacterium Geobacillus stearothermophilus T-1. Gan1D has been shown to exhibit bifunctional activity, possessing both 6-phospho-ß-galactosidase and 6-phospho-ß-glucosidase activities. We report herein the complete 3D crystal structure of Gan1D, together with its acid/base catalytic mutant Gan1D-E170Q. The tertiary structure of Gan1D conforms well to the (ß/α)8 TIM-barrel fold commonly observed in GH enzymes, and its quaternary structure adopts a dimeric assembly, confirmed by gel-filtration and small-angle X-ray scattering results. We present also the structures of Gan1D in complex with the putative substrate cellobiose-6-phosphate (Cell6P) and the degradation products Glc6P and Gal6P. These complexes reveal the specific enzyme-substrate and enzyme-product binding interactions of Gan1D, and the residues involved in its glycone, aglycone, and phosphate binding sites. We show that the different ligands trapped in the active sites adopt different binding modes to the protein, providing a structural basis for the dual galactosidase/glucosidase activity observed for this enzyme. Based on this information, specific mutations were performed on one of the active site residues (W433), shifting the enzyme specificity from dual activity to a significant preference toward 6-phospho-ß-glucosidase activity. These data and their comparison with structural data of related glucosidases and galactosidases are used for a more general discussion on the structure-function relationships in this sub-group of GH1 enzymes. DATABASES: Atomic coordinates of Gan1D-wild-type (WT)-P1, Gan1D-WT-C2, Gan1D-E170Q, Gan1D-WT-Gal6P, Gan1D-WT-Glc6P, and Gan1D-E170Q-Cell6P have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, under accession codes 5OKB, 5OKJ/5OKH, 5OKA/5OK7, 5OKQ/5OKK, 5OKS/5OKR, and 5OKG/5OKE, respectively.

Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum.

Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative σI factors has been demonstrated in Clostridium (Ruminiclostridium) thermocellum In the present report, we provide experimental evidence demonstrating that the C. thermocellum cipA gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative σI factors and by the vegetative σA factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of C. thermocellum cipA are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved σI- and σA-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, Clostridium straminisolvens, Clostridium clariflavum, Acetivibrio cellulolyticus, and Clostridium sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of Clostridium cellulovorans matches the predicted σI-dependent promoter identified in the present work rather than the previously proposed σA promoter. With the exception of C. cellulovorans, both σI and σA promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5'-untranslated regions (5'-UTRs). Furthermore, these 5'-UTRs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components.IMPORTANCE Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of C. thermocellum and related cellulosome-producing bacteria en route to the consolidated bioprocessing of biomass.

A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40-150 µg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

Decoding Biomass-Sensing Regulons of Clostridium thermocellum Alternative Sigma-I Factors in a Heterologous Bacillus subtilis Host System.

The Gram-positive, anaerobic, cellulolytic, thermophile Clostridium (Ruminiclostridium) thermocellum secretes a multi-enzyme system called the cellulosome to solubilize plant cell wall polysaccharides. During the saccharolytic process, the enzymatic composition of the cellulosome is modulated according to the type of polysaccharide(s) present in the environment. C. thermocellum has a set of eight alternative RNA polymerase sigma (σ) factors that are activated in response to extracellular polysaccharides and share sequence similarity to the Bacillus subtilis σI factor. The aim of the present work was to demonstrate whether individual C. thermocellum σI-like factors regulate specific cellulosomal genes, focusing on C. thermocellum σI6 and σI3 factors. To search for putative σI6- and σI3-dependent promoters, bioinformatic analysis of the upstream regions of the cellulosomal genes was performed. Because of the limited genetic tools available for C. thermocellum, the functionality of the predicted σI6- and σI3-dependent promoters was studied in B. subtilis as a heterologous host. This system enabled observation of the activation of 10 predicted σI6-dependent promoters associated with the C. thermocellum genes: sigI6 (itself, Clo1313_2778), xyn11B (Clo1313_0522), xyn10D (Clo1313_0177), xyn10Z (Clo1313_2635), xyn10Y (Clo1313_1305), cel9V (Clo1313_0349), cseP (Clo1313_2188), sigI1 (Clo1313_2174), cipA (Clo1313_0627), and rsgI5 (Clo1313_0985). Additionally, we observed the activation of 4 predicted σI3-dependent promoters associated with the C. thermocellum genes: sigI3 (itself, Clo1313_1911), pl11 (Clo1313_1983), ce12 (Clo1313_0693) and cipA. Our results suggest possible regulons of σI6 and σI3 in C. thermocellum, as well as the σI6 and σI3 promoter consensus sequences. The proposed -35 and -10 promoter consensus elements of σI6 are CNNAAA and CGAA, respectively. Additionally, a less conserved CGA sequence next to the C in the -35 element and a highly conserved AT sequence three bases downstream of the -10 element were also identified as important nucleotides for promoter recognition. Regarding σI3, the proposed -35 and -10 promoter consensus elements are CCCYYAAA and CGWA, respectively. The present study provides new clues for understanding these recently discovered alternative σI factors.

Structure-function relationships in Gan42B, an intracellular GH42 ß-galactosidase from Geobacillus stearothermophilus.

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 ß-galactosidase capable of hydrolyzing short ß-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Šresolution) and its catalytic mutant E323A (at 2.50 Šresolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/ß domain, and the smallest all-ß domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Šfrom each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Šat the wide opening and ∼5 Šat the small opening and ∼40 Šin length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure-activity aspects in this GH family.

Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: structural and functional significance of the intermodular linker.

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60-70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.

Three cellulosomal xylanase genes in Clostridium thermocellum are regulated by both vegetative SigA (σ(A)) and alternative SigI6 (σ(I6)) factors.

Clostridium thermocellum efficiently degrades crystalline cellulose by a high molecular weight protein complex, the cellulosome. The bacterium regulates its cellulosomal genes using a unique extracellular biomass-sensing mechanism that involves alternative sigma factors and extracellular carbohydrate-binding modules attached to intracellular anti-sigma domains. In this study, we identified three cellulosomal xylanase genes that are regulated by the σ(I6)/RsgI6 system by utilizing sigI6 and rsgI6 knockout mutants together with primer extension analysis. Our results indicate that cellulosomal genes are expressed from both alternative σ(I6) and σ(A) vegetative promoters.

Preliminary crystallographic analysis of Xyn52B2, a GH52 ß-D-xylosidase from Geobacillus stearothermophilus T6.

Geobacillus stearothermophilus T6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of ß-xylosidases. One of these ß-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombic P212121 space group, with average unit-cell parameters a = 97.7, b = 119.1, c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Šresolution), the E335G catalytic mutant (2.95 Šresolution), a potential mercury derivative (2.15 Šresolution) and a selenomethionine derivative (3.90 Šresolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

Structure-specificity relationships in Abp, a GH27 ß-L-arabinopyranosidase from Geobacillus stearothermophilus T6.

L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system in Geobacillus stearothermophilus T6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular ß-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove ß-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Šresolution) and its catalytic mutant Abp-D197A with (at 2.20 Šresolution) and without (at 2.30 Šresolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-ß domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Šfrom each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer made up of two `open-pincers' dimers, which clamp around each other to form a central cavity. The four active sites of the Abp tetramer are situated on the inner surface of this cavity, all opening into the central space of the cavity. The biological relevance of this tetrameric structure is supported by independent results obtained from size-exclusion chromatography (SEC), dynamic light-scattering (DLS) and small-angle X-ray scattering (SAXS) experiments. These data and their comparison to the structural data of related GH27 enzymes are used for a more general discussion concerning structure-selectivity aspects in this glycoside hydrolase (GH) family.

Cloning, purification and preliminary crystallographic analysis of Ara127N, a GH127 ß-L-arabinofuranosidase from Geobacillus stearothermophilus T6.

The L-arabinan utilization system of Geobacillus stearothermophilus T6 is composed of five transcriptional units that are clustered within a 38 kb DNA segment. One of the transcriptional units contains 11 genes, the last gene of which (araN) encodes a protein, Ara127N, that belongs to the newly established GH127 family. Ara127N shares 44% sequence identity with the recently characterized HypBA1 protein from Bifidobacterium longum and thus is likely to function similarly as a ß-L-arabinofuranosidase. ß-L-Arabinofuranosidases are enzymes that hydrolyze ß-L-arabinofuranoside linkages, the less common form of such linkages, a unique enzymatic activity that has been identified only recently. The interest in the structure and mode of action of Ara127N therefore stems from its special catalytic activity as well as its membership of the new GH127 family, the structure and mechanism of which are only starting to be resolved. Ara127N has recently been cloned, overexpressed, purified and crystallized. Two suitable crystal forms have been obtained: one (CTP form) belongs to the monoclinic space group P21, with unit-cell parameters a = 104.0, b = 131.2, c = 107.6 Å, ß = 112.0°, and the other (RB form) belongs to the orthorhombic space group P212121, with unit-cell parameters a = 65.5, b = 118.1, c = 175.0 Å. A complete X-ray diffraction data set has been collected to 2.3 Šresolution from flash-cooled crystals of the wild-type enzyme (RB form) at -173°C using synchrotron radiation. A selenomethionine derivative of Ara127N has also been prepared and crystallized for multi-wavelength anomalous diffraction (MAD) experiments. Crystals of selenomethionine Ara127N appeared to be isomorphous to those of the wild type (CTP form) and enabled the measurement of a three-wavelength MAD diffraction data set at the selenium absorption edge. These data are currently being used for detailed three-dimensional structure determination of the Ara127N protein.

Enzymatically synthesized dextran nanoparticles and their use as carriers for nutraceuticals.

This study evaluated the use of enzymatically synthesized dextran nanoparticles to entrap a hydrophobic nutraceutical, the isoflavone genistein. The focus was on the synthesis of pure dextran as the material for the entire nanoparticle, and their inclusion-complexation of genistein. Under optimal conditions (pH 5.2-6 and sucrose concentration >0.5 M), dextransucrase generated spherical dextran nanoparticles (100-450 nm). The two nutraceutical inclusion methods were DMSO dilution in water and acidification. Optimization of the inclusion processes produced nanosized dextran particles containing genistein. The DMSO method was found to be more suitable for inclusion of genistein in dextran, resulting in a higher genistein load (5.6 ± 0.1 g genistein per 100 g particles), and a higher percentage of nanosized particles (85%, 105-400 nm). For both protocols, addition of a freeze-drying step exerted a positive effect presumably due to the formation of new hydrogen bonds and van der Waals interactions.

Multiple regulatory mechanisms control the expression of the Geobacillus stearothermophilus gene for extracellular xylanase.

Geobacillus stearothermophilus T-6 produces a single extracellular xylanase (Xyn10A) capable of producing short, decorated xylo-oligosaccharides from the naturally branched polysaccharide, xylan. Gel retardation assays indicated that the master negative regulator, XylR, binds specifically to xylR operators in the promoters of xylose and xylan-utilization genes. This binding is efficiently prevented in vitro by xylose, the most likely molecular inducer. Expression of the extracellular xylanase is repressed in medium containing either glucose or casamino acids, suggesting that carbon catabolite repression plays a role in regulating xynA. The global transcriptional regulator CodY was shown to bind specifically to the xynA promoter region in vitro, suggesting that CodY is a repressor of xynA. The xynA gene is located next to an uncharacterized gene, xynX, that has similarity to the NIF3 (Ngg1p interacting factor 3)-like protein family. XynX binds specifically to a 72-bp fragment in the promoter region of xynA, and the expression of xynA in a xynX null mutant appeared to be higher, indicating that XynX regulates xynA. The specific activity of the extracellular xylanase increases over 50-fold during early exponential growth, suggesting cell density regulation (quorum sensing). Addition of conditioned medium to fresh and low cell density cultures resulted in high expression of xynA, indicating that a diffusible extracellular xynA density factor is present in the medium. The xynA density factor is heat-stable, sensitive to proteases, and was partially purified using reverse phase liquid chromatography. Taken together, these results suggest that xynA is regulated by quorum-sensing at low cell densities.