Computational and SAXS-based structure insights of pectin acetyl esterase (CtPae12B) of family 12 carbohydrate esterase from Clostridium thermocellum ATCC 27405.

Pectin is a complex form of polysaccharide and is composed of several structural components that require the concerted action of several pectinases for its complete degradation. In this study, in silico and solution structure of a pectin acetyl esterase (CtPae12B) of family 12 carbohydrate esterase (CE12) from Clostridium thermocellum was determined. The CtPae12B modelled structure, showed a new α/ß hydrolase fold, similar to the fold found in the crystal structures of its nearest homologues from CE12 family, which differed from α/ß hydrolase fold found in glycoside hydrolases. In the active site of CtPae12B, two loops (loop1 and loop6) play an important role in the formation of a catalytic triad Ser15-Asp187-His190, where Ser15 acts as a nucleophile. The structural stability of CtPae12B and its catalytic site was detected by performing molecular dynamic (MD) simulation which showed stable and compact conformation of the structure. Molecular docking method was employed to analyse the conformations of various suitable ligands docked at the active site of CtPae12B. The stability and structural specificity of the catalytic residues with the ligand, 4-nitrophenyl acetate (4-NPA) was confirmed by MD simulation of CtPae12B-4NPA docked complex. Moreover, it was found that the nucleophile Ser15, forms hydrophobic interaction with 4-NPA in the active site to complete covalent catalysis. Small angle X-ray scattering analysis of CtPae12B at 3 mg/mL displayed elongated, compact and monodispersed nature in solution. The ab initio derived dummy model showed that CtPae12B exists as a homotrimer at 3 mg/mL which was also confirmed by dynamic light scattering.Communicated by Ramaswamy H. Sarma.

Magnetic Separation of Elastin-like Polypeptide Receptors for Enrichment of Cellular and Molecular Targets.

Protein-conjugated magnetic nanoparticles (mNPs) are promising tools for a variety of biomedical applications, from immunoassays and biosensors to theranostics and drug-delivery. In such applications, conjugation of affinity proteins (e.g., antibodies) to the nanoparticle surface many times compromises biological activity and specificity, leading to increased reagent consumption and decreased assay performance. To address this problem, we engineered a biomolecular magnetic separation system that eliminates the need to chemically modify nanoparticles with the capture biomolecules or synthetic polymers of any kind. The system consists of (i) thermoresponsive magnetic iron oxide nanoparticles displaying poly(N-isopropylacrylamide) (pNIPAm), and (ii) an elastin-like polypeptide (ELP) fused with the affinity protein Cohesin (Coh). Proper design of pNIPAm-mNPs and ELP-Coh allowed for efficient cross-aggregation of the two distinct nanoparticle types under collapsing stimuli, which enabled magnetic separation of ELP-Coh aggregates bound to target Dockerin (Doc) molecules. Selective resolubilization of the ELP-Coh/Doc complexes was achieved under intermediate conditions under which only the pNIPAm-mNPs remained aggregated. We show that ELP-Coh is capable of magnetically separating and purifying nanomolar quantities of Doc as well as eukaryotic whole cells displaying the complementary Doc domain from diluted human plasma. This modular system provides magnetic enrichment and purification of captured molecular targets and eliminates the requirement of biofunctionalization of magnetic nanoparticles to achieve bioseparations. Our streamlined and simplified approach is amenable for point-of-use applications and brings the advantages of ELP-fusion proteins to the realm of magnetic particle separation systems.

Molecular determinants of substrate specificity revealed by the structure of Clostridium thermocellum arabinofuranosidase 43A from glycosyl hydrolase family 43 subfamily 16.

The recent division of the large glycoside hydrolase family 43 (GH43) into subfamilies offers a renewed opportunity to develop structure-function studies aimed at clarifying the molecular determinants of substrate specificity in carbohydrate-degrading enzymes. α-L-Arabinofuranosidases (EC 3.2.1.55) remove arabinose side chains from heteropolysaccharides such as xylan and arabinan. However, there is some evidence suggesting that arabinofuranosidases are substrate-specific, being unable to display a debranching activity on different polysaccharides. Here, the structure of Clostridium thermocellum arabinofuranosidase 43A (CtAbf43A), which has been shown to act in the removal of arabinose side chains from arabinoxylan but not from pectic arabinan, is reported. CtAbf43A belongs to GH43 subfamily 16, the members of which have a restricted capacity to attack xylans. The crystal structure of CtAbf43A comprises a five-bladed ß-propeller fold typical of GH43 enzymes. CtAbf43A displays a highly compact architecture compatible with its high thermostability. Analysis of CtAbf43A along with the other member of GH43 subfamily 16 with known structure, the Bacillus subtilis arabinofuranosidase BsAXH-m2,3, suggests that the specificity of subfamily 16 for arabinoxylan is conferred by a long surface substrate-binding cleft that is complementary to the xylan backbone. The lack of a curved-shaped carbohydrate-interacting platform precludes GH43 subfamily 16 enzymes from interacting with the nonlinear arabinan scaffold and therefore from deconstructing this polysaccharide.

A useful method integrating production and immobilization of recombinant cellulase.

The development of cellulase-based bioprocess is afflicted by the processing efficiency of enzymes. To address this issue, a method based on artificial oil bodies (AOBs) was proposed to integrate production and immobilization of recombinant cellulase. First, the heterologous endoglucanase (celA), cellobiohydrolase (celK), and ß-glucosidase (gls) genes were individually fused with oleosin, a structural protein of plant seed oils. After expression in Escherichia coli, each fusion protein of insolubility was mixed together with plant oils. AOBs were assembled by subjecting the mixture to sonication. Consequently, active CelA, CelK, and Gls were resumed and co-immobilized on AOBs surface. Finally, the assembly condition (including the protein ratio) and the reaction condition were further optimized by response surface methodology. The resulting AOBs-bound cellulase remained stable for 4 cycles of cellulose-hydrolyzed reactions. Overall, the result shows a promise of this proposed approach for processing recombinant cellulase, which may provide a facile method to investigate optimum combination of cellulase components towards various cellulosic materials.

Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction.

Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module.

Mechanism of bacterial cell-surface attachment revealed by the structure of cellulosomal type II cohesin-dockerin complex.

Bacterial cell-surface attachment of macromolecular complexes maintains the microorganism in close proximity to extracellular substrates and allows for optimal uptake of hydrolytic byproducts. The cellulosome is a large multienzyme complex used by many anaerobic bacteria for the efficient degradation of plant cell-wall polysaccharides. The mechanism of cellulosome retention to the bacterial cell surface involves a calcium-mediated protein-protein interaction between the dockerin (Doc) module from the cellulosomal scaffold and a cohesin (Coh) module of cell-surface proteins located within the proteoglycan layer. Here, we report the structure of an ultra-high-affinity (K(a) = 1.44 x 10(10) M(-1)) complex between type II Doc, together with its neighboring X module from the cellulosome scaffold of Clostridium thermocellum, and a type II Coh module associated with the bacterial cell surface. Identification of X module-Doc and X module-Coh contacts reveal roles for the X module in Doc stability and enhanced Coh recognition. This extremely tight interaction involves one face of the Coh and both helices of the Doc and comprises significant hydrophobic character and a complementary extensive hydrogen-bond network. This structure represents a unique mechanism for cell-surface attachment in anaerobic bacteria and provides a rationale for discriminating between type I and type II Coh modules.