Kedar Sharma

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 β-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.

Low-resolution SAXS and comparative modeling based structure analysis of endo-β-1,4-xylanase a family 10 glycoside hydrolase from Pseudopedobacter saltans comb. nov.

The structure and biophysical properties of endo β-1,4-xylanase (PsGH10A) of family 10 glycoside hydrolase were characterized. The modeled PsGH10A structure showed classical (β/α)8-barrel fold. Ramachandran plot displayed 99.1% residues in favored and 0.3% in the generously allowed region and only 0.6% residues in disallowed region. The secondary structure analysis of PsGH10A by CD revealed 31.75% α-helices 20.0% β-strands and 48.25% random coils. Protein melting study of PsGH10A showed complete unfolding at 60°C and did not require any metal ion for its stability. Structural superposition and docking analysis confirmed the involvement of Glu156 and Glu263 residues in catalysis. SAXS analysis displayed that PsGH10A is monomeric in nature showing fully folded state in solution form. Guinier analysis gave the radius of gyration (Rg) 2.23-2.29nm. Kratky plot indicated that the protein is fully folded globular shaped and flexible in solution form. The ab initio derived dummy model of PsGH10A displayed chicken thigh like shape. The ab initio derived dummy model superposed well with its comparative modeled structure except the N-terminal His-tag region.

Insights into structure and reaction mechanism of β-mannanases.

β-mannanases have been shown to play an important role in various biological processes such as the cell wall component degradation, defence signalling in plants, the mobilization of storage reserves and in various industrial processes. To date, glycoside hydrolases (GHs) have been divided into 135 families and 14 clans from A to N based upon their sequence, overall structural fold and function. β -mannanases belong glycoside hydrolases and exist under four different glycoside hydrolase families, GH5, GH26, GH113 and GH134. GH5 and GH26 are combined in clan GH-A. GH5 and GH26 contain hydrolases which follow the retaining type reaction mechanism. Structural survey of β- mannanases of GH5 and GH26, suggests that both families contain similar TIM barrel fold. In addition, they also share common catalytic residues and their location in the structure. Despite these structural similarities, a distinct difference lies between the substrate binding sub-sites which define substrate specificity. This review summarizes the recent reports on the structure and function perspectives of β- mannanases of GH5 and GH26 and highlights the similarities and differences between them.

7 – Proteolytic Enzymes

Proteolytic enzymes, also known as “proteases,” cleave the peptide bonds that connect two amino acids. They follow a hydrolytic reaction mechanism. Proteolytic enzymes are produced by both prokaryotic and eukaryotic organisms in which they perform key biological functions. Proteolytic enzymes have great commercial value as they are in demand from food, dairy, detergent, and leather-processing industries. Proteolytic enzymes have also emerged as therapeutics and several protease-based therapies have been approved. Microorganisms are a major source of commercial proteolytic enzymes because of high productivity and the ease of purification of the enzymes. Major challenges for research in the commercial exploitation of proteases are engineering new specificities, their stability, and their use as therapeutics.

Comparative analysis of pretreatment methods on sorghum (Sorghum durra) stalk agrowaste for holocellulose content

Abstract This study compares different types of pretreatment methods, such as thermal pretreatment at 120 °C, autoclaving, microwaving and ultrasonication in the presence of water, dilute acid (1% H2SO4) or dilute alkali (1% NaOH) on Sorghum stalk with respect to the holocellulose and Acid Detergent/Insoluble Lignin content. Among all the methods, pretreatment with 1% NaOH along with autoclaving at 121 °C and 15 psi for 30 min was the most effective method for Sorghum stalk. Fourier Transform Infra-Red spectroscopy analysis of this pretreated biomass showed the removal of lignin and Field Emission Scanning Electron Microscope analysis displayed enhanced surface roughness. The enzymatic hydrolysis of raw and best pretreated Sorghum stalk using recombinant endo-β-1,4-glucanase (CtCel8A) and β-1,4-glucosidase (CtBgl1A) both from Clostridium thermocellum gave glucose yields, 22.4 mg/g raw biomass and 34 mg/g pretreated biomass, respectively, resulting in 1.5-fold increase of glucose yield after the pretreatment.

Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase

The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

SAXS and homology modelling based structure characterization of pectin methylesterase a family 8 carbohydrate esterase from Clostridium thermocellum ATCC 27405

Pectin methylesterase (CtPME) from Clostridium thermocellum of family 8 carbohydrate esterase (CE8) belongs to pectin methylesterase super family (E.C.3.1.1.11). BLAST analysis of CtPME showed 38% sequence identity with PME from Erwinia chrysanthemi. Multiple sequence alignment of CtPME with other known structures of pectin methylesterase revealed the conserved and semi-conserved amino acid residues. Homology modelling of CtPME structure revealed a characteristic right handed parallel β-helices. The energy of modelled structure was minimized by using YASARA software. The Ramachandran plot of CtPME shows 83.7% residues in non-glycine and non-proline residues in most-favorable region, 13.8% in additional allowed region and 1.4% in generously allowed region, indicating that CtPME has a stable conformation. The secondary structure of CtPME predicted using PSI-Pred software and confirmed by the circular dichroism (CD) showed α-helices (3.1%), β-sheets (40.1%) and random coils (56.9%). Small Angle X-ray Scattering (SAXS) analysis demonstrated the overall shape and structural characterization of CtPME in solution form. Guinier analysis gave the radius of gyration (Rg) 2.28 nm for globular shape and 0.74 nm for rod shape. Kratky plot gave the indication that protein is fully folded in solution. The ab initio derived dummy atom model of CtPME superposed well on modelled CtPME structure.

The multi-ligand binding first family 35 Carbohydrate Binding Module (CBM35) of Clostridium thermocellum targets rhamnogalacturonan I

Carbohydrate Binding Modules (CBMs) targeting cellulose, xylan and mannan have been reported, however, a CBM targeting rhamnogalacturonan I (RG I) has never been identified. We had studied earlier a rhamnogalacturonan lyase (CtRGL) from Clostridium thermocellum that was associated with a family 35 CBM, Rgl-CBM35. In this study we show that Rgl-CBM35 displays binding with β-d-glucuronic acid (β-D-GlcpA), Δ4,5-anhydro-d-galactopyranosyluronic acid (Δ4,5-GalpA), rhamnogalacturonan I, arabinan, galactan, glucuronoxylans and arabinoxylans. Rgl-CBM35 contains a conserved ligand binding site in the loops known for binding β-D-GlcpA and Δ4,5-GalpA moiety of unsaturated RG I and pectic-oligosaccharides. Mutagenesis revealed that Asn118 plays an important role in binding β-D-GlcpA, Δ4,5-GalpA, sugarbeet arabinan and potato galactan at its conserved ligand binding site present in surface exposed loops. EDTA-treated Rgl-CBM35 showed no affinity towards β-D-GlcpA and Δ4,5-GalpA underscoring Ca2+ mediated ligand recognition. Contrastingly, the EDTA-treated Rgl-CBM35 and its mutant N118A displayed affinity for sugarbeet arabinan and potato galactan. The curtailed affinity of Y37A/N118A and R69A/N118A double mutants towards sugarbeet arabinan emphasized the presence of a second ligand binding site. Rgl-CBM35 is the first CBM reported to primarily target RG I and also is the first member of family 35 CBM possessing at least two ligand binding sites.

Insights into the structural characteristics and substrate binding analysis of chondroitin AC lyase (PsPL8A) from Pedobacter saltans

The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α)6 incomplete toroidal fold and a layered β sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% β sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×105) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during β-elimination.

Selection of early segregating progeny lines of chickpea (Cicer arietinum L.) for high yield under terminal drought stress conditions

Drought is one of the most devastating abiotic stresses, spreading around the world and limiting the productivity of chickpea. The present study was carried out to select the high yielding progeny lines of chickpea to combat terminal drought stress conditions. Parameters that were measured included phenological traits; yield and yieldrelated traits viz. number of branches plant, number of pods plant, number of seeds pod, 100 seed weight and biological yield in F3 progeny lines of cross HC-1 × RSG 931 along with parental chickpea genotypes. The data was analyzed using statistical program and all yield-related traits were found to be positively correlated with seed yield. Four progeny lines viz. P9, P15, P17 and P18 had higher seed yield plant than drought tolerant parental chickpea genotype, RSG 931 under terminal drought stress conditions. These superior progeny lines could be incorporated in chickpea breeding program to increase yield under terminal drought stress.