In the same vein, I would like to thank my research group members Aruna, Arun, Rwivoo, Vikky, Kedar and Ashutosh for their help and maintaining a pleasant working environment in the lab. In the present study the 3-dimensional structural features of the catalytic module CtXyn30A of the GH30 family and the associated module CtCBM6 of the family 6 from Clostridium thermocellum were characterized by using a computational approach and X-ray crystallography technique to understand the architect of the substrate binding cleft in Ct general structure feature of CtXyn30A.
General Introduction 1
Structural polysaccharides in plants
- Cellulose
- Hemicellulose
- Xylans
- Lignins
- Pectins
- Homogalacturonan
- Heterogalacturonan
Homogalacturonan (HG) is composed of linear chain of α-D-galactosyluron residues linked by α-(1→4) glycosidic bonds with some esterification at the carboxylic acids of the galacturonates by methanol (methyl esterified) or L-rhamnose ( α -6-deoxyhexose) residues (Fig. 1.11) (Ridley et al., 2001). Rhamnogalacturonan-II (RG-II) is a structurally complex pectic polysaccharide with a main chain backbone of 1,4-linked α-D-galacturonic acid (GalA) residues (O'Neill et al., 2004).
Carbohydrate-active enzymes
- Glycosyltransferases
- Polysaccharide lyases
- Carbohydrate esterases
- Glycoside hydrolases
- Modular nature of glycoside hydrolases
- Mechanism of action of Glycoside hydrolase
- Family 30 glycoside hydrolase
- GH30 sub-family 8 and CtXynGH30
- Applications of glucuronoxylan-xylanohydrolase
41% of the total CAZymes at present and is practically present in every single organism (Cantarel et al., 2009). The enzyme glycoside hydrolase glucuronoxylan-xylanohydrolase can be described as a xylanase enzyme which cleaves the β-(1→4) bonds of 4-O-methylglucuronoxylan (MeGXn) as directed by the position along the xylan chain of an α-(1→2) ). 4-O-methylglucuronate (MeGA) bound moiety (St John et al., 2010).
Carbohydrate binding modules
- Classification of carbohydrate binding modules
- Functions of carbohydrate binding modules
- Applications of carbohydrate binding modules
- Carbohydrate Binding Modules of family 6
The major application of CBMs involves the construction of an expression vector containing a CBM as a fusion tag (Novy et al., 1997). CBMs are used in immobilized affinity ligand technology for the purification of biomolecules (Greenwood et al., 1992).
The microorganism
Cellulosomes …
Glycerol stocks were prepared from E. coli BL-21 cells harboring recombinant plasmids and maintained at -80ºC. The cells in 100 ml of LB medium were cultured for enzyme purification. The resolution was reduced to a conservative 1.4 Å (the CC1/2 value is lower than 0.5 for higher resolution data).
Significance of the investigation
Objectives of the present study
- Specific Objectives
The reaction conditions for the enzyme assay, such as optimal pH, temperature and buffer ionic strength, will be optimized. Enzyme activity with various substrates including synthetic pNP (para nitro phenyl) glycosides will be discussed.
Cloning, expression and purification of glucuronoxylan-
Materials and methods
- Chemicals, Reagents and kits
- Microorganisms
- PCR amplification of gene encoding for CtXynGH30 and
- Agarose gel electrophoresis of PCR amplified and other DNA
- DNA loading dye
- Extraction of DNA from agarose gel
- DNA gel extraction protocol
- Cloning of PCR Products into a pGEM-T Easy Vector
- Restriction enzyme digestion of the pGEM-T Easy clone
- Generation of pET-28a(+) expression vector for cloning of genes
- Ligation of inserts released from recombinant pGEM-T Easy
- Preparation of E. coli (DH5α) competent cells …
- Preparation of Luria-Bertani (LB) medium
- Preparation of LB-agar medium
- Preparation of SOC medium
- Transformation of ligated DNA using E. coli (DH5α) cells
- Isolation of recombinant plasmid DNA
- Plasmid miniprep (alkaline lysis) protocol
- Screening of recombinant plasmid DNAs for positive clones
- Preparation of E. coli (BL-21) competent cells
- Isolation of recombinant plasmid DNA
- Plasmid isolation protocol (Sigma-Aldrich)
- Transformation of plasmid DNA using E. coli BL-21 cells
- Overexpression of recombinant proteins
- Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis
- Preparation of SDS-PAGE gel
- Preparation of acrylamide 30% (w/v) solution
- Polymerization of SDS-PAGE gel
- Preparation of SDS-PAGE running buffer
- Preparation of sample buffer
- Preparation of staining and destaining solutions
- Purification of recombinant proteins
- Protein estimation by Bradford method
- Preparation of Bradford reagent
The supernatant was carefully removed and the pellet was gently resuspended in 3.0 ml of sterile ice-cold 0.1 M CaCl2 solution. Ethanol was again removed by micropipette and the microcentrifuge tube containing plasmid DNA was air-dried for 5 min. 10 µl of RNAseA solution was added (20 µg/ml final concentration) and the plasmid DNA was mixed well and stored at -20°C.
The cells were then centrifuged at 13000 g for 1 minute and the process was repeated with an additional 1.5 ml of culture. 200 μl of lysis solution was added to each tube and the tubes were gently inverted 5–6 times. The SDS-PAGE gels were run using a 1x running or tank buffer prepared from the 5x stock solution as described in Table 2.2.13. w/v) SDS was added and the final volume was adjusted to 1 liter.
A 5-fold sample loading buffer was prepared by dissolving the components, maintaining the concentration of the components as described in Table 2.2.14, and the pH of the buffer was adjusted to 6.8.
Results and Discussion
- Defining boundaries to modular full length CtXynGH30
- PCR amplification of CtXynGH30, CtXyn30A and CtCBM6
- Cloning of PCR products into a pGEM-T Easy vector
- Cloning and expression of CtXynGH30, CtXyn30A and CtCBM6 78
- Isolation of plasmid DNA from colonies
- Screening of plasmid DNAs for positive clones
- Sequencing of positive recombinant plasmid DNAs
- Hyper-expression analysis of recombinant proteins
- Purification of CtXynGH30, CtXyn30A and CtCBM6
- Purification profile of CtXynGH30
- Purification profile of CtXyn30A
- Purification profile of CtCBM6
- Protein estimation of expressed recombinant proteins …
In the present study, the purified recombinant proteins, CtXynGH30 and CtXyn30A modules were biochemically and functionally characterized. Zymogram analysis of CtXynGH30 and CtXyn30A was performed in the presence of beech wood xylan to confirm xylanase activity following the method as previously described by Ghosh et al., (2013). The effect of metal ions and chemical agents on the activity of CtXynGH30 and CtXyn30A was studied at low (1 mM) as well as high (10 mM) concentrations as mentioned in Table 3.3.2.
The complete methodology for cloning, expression and purification of CtXyn30A was described in detail in Chapter 2. The last round of refinement for structures with PDB codes 4uqe, 4uqd, 4uqc and 4uqb was performed in the PDBredo web server (Joosten et al. , 2012). They are important in binding various small molecules, as shown in Figures 4.3.16 and 4.3.17.
Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Higgins D.J. CLUSTAL_X Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. 2004) The REFMAC5 dictionary: the organization of prior chemical knowledge and guidance for its use.
Conclusions
Biochemical and functional characterization of catalytic
Materials and Methods
- Substrates and Chemical
- Cloning, over-expression and purification of CtXynGH30 and
- Enzyme activity assay
- Preparation of reagents for reducing sugar estimation
- Generation of standard plot of D -xylose
- Calculation of enzyme activity
- Assay of CtXynGH30 and CtXyn30A with natural and
- Substrate specificity of CtXynGH30 and CtXyn30A on a
- Activity staining of CtXynGH30 and CtXyn30A
- Determination of optimum pH and pH stability of CtXynGH30
- Temperature optima and thermal stability of CtXynGH30 and
CtXynGH30 and CtXyn30A were tested against various natural polysaccharides to determine substrate specificity. The concentration of reducing sugar was estimated using a standard curve of xylose, as CtXynGH30 and CtXyn30A showed maximum activity against the substrates with xylose residues in the main chain. The thermal stability of CtXynGH30 and CtXyn30A was determined by incubating 50 μl (85 μg) of each enzyme at different temperatures for 30 min.
The effect of different metal ions and chemical agents on the activity of CtXynGH30 and CtXyn30A was analyzed. The maximum specific activity of CtXynGH30 and CtXyn30A was found to be 31 and 34 U/mg, respectively, with beech xylan (Table 3.3.1). The activity of CtXynGH30 and CtXyn30A was not significantly affected by Mg2+, Ca2+ and β-mercaptoethanol.
The substrate specificity analysis of CtXynGH30 and CtXyn30A clearly showed that they act on xylan-based polysaccharides with xylose as a monomeric unit.
Introduction
The presence of these acetyl groups is responsible for the partial solubility of xylan in water (Anna Ebringerova, 2000; Larson et al., 2003). Glucuronoxylan xylanohydrolase requires this methyl glucuronic acid or glucuronic acid substitution to act on the main chain xylan, effectively breaking down the acidic substituted xylans. Briefly: protein XynA from Erwinia chrysanthemi (Larson et al., 2003) and XynC from Bacillus subtilis (St John et al., 2009).
Both preferentially hydrolyze glucuronoxylan to branched xylo-oligosaccharides and showed negligible activity on linear β-1-4-linked xylo-oligosaccharides. Two more structures of xylanase of the same subfamily have recently been solved, viz. from Clostridium papyrosolvens (CpC71), which showed low specificity for glucuronoxylan (St John et al. 2014) and Xyn30D from P. To our knowledge, there is no structural and biochemical report available on the glucuronoxylan-xylanohydrolase enzyme from clostridium thermocellum.
This study describes the insight into the structural aspect of CtXyn30A, which represents the first glucuronoxylan xylanohydrolase from C.
Materials and Methods
- Computational and molecular docking analyses of CtXyn30A …
- Homology modeling
- Model refinement and quality assessment
- Molecular dynamics
- Prediction of active site
- Docking study on modeled CtXyn30A
- Cloning, crystallization and structural analysis of CtXyn30A
- Cloning, expression and purification of CtXyn30A
- Crystallization conditions for CtXyn30A
- Data collection, structure determination and refinement of
A Ramachandran plot, which graphically represents the combination of possible and allowed torsion angles, backbone phi (ϕ) and psi (ψ) dihedral angles, was obtained using the PROCHECK program (Laskowski, et al., 1993). The alignment was generated by the program Clustal_X (Thompson et al., 1997) and the figure was produced by ESpript (http://espript.ibcp.fr). The Lamarckian Genetic Algorithm (LGA) was implemented for docking simulation and conformational search (Morris, et al., 1998).
CtXyn30A was crystallized under several conditions and data from all the different crystals obtained were collected at beamline ID29 at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) (de Sanctis et al., 2012), Diamond Light Source (Harwell, UK) and at PROXIMA-1 at SOLEIL (Orsay, France) using a PILATUS 6M detector with the crystals cooled to 100 K using a Cryostream (Oxford Cryosystems) (Verma et al., 2013). A model comprising 1 chain of 389 residues (native protein plus 3 residues at the N terminus due to the cloning construct) was constructed from the initial map with the COOT program ( Emsley et al., 2010 ). The final model was verified with PROCHECK (Laskowski, et al., 1993) and checked and validated during submission to the PDB.
Visualization and analysis of structures and figure generation were performed using PyMOL (Schrodinger, 2010) and UCSF Chimera (Pettersen et al., 2004).
Results and Discussion
- In silico structure analysis of CtXyn30A
- Homology modeling and structure validation of
- Molecular dynamics simulation of CtXyn30A
- Overall structural features of CtXyn30A
- Catalytic core of CtXyn30A
- Probable mechanism of catalysis
- Docking of substrate at the active site
- Crystallization and 3-dimensional structure analysis of native
- Crystals of CtXyn30A under different conditions
- Three dimensional structure analysis of CtXyn30A
- CtXyn30A catalytic site analysis
CtXyn30A structure consists of the expected (β/α)8 TIM barrel which is classified as belonging to the carbohydrate-active enzyme database strain A enzymes (4/7 hydrolases). This loop in CtXyn30A is similar in XynC and CtXyn30A, which is consistent with the conservation of the residues observed in this region in the three structures (Fig. 4.3.13). Surface representation of CtXyn30A (A, D), XynC (B, E) and XynA (C, F) catalytic site (PDBs id, 4UQD, 3KL0 and 2Y24, respectively) with the various ligands observed, represented in sticks (A, B) and C, respectively), (PDBs id, 4CKQ, 4UQB, 4UQC, 4UQD for CtXyn30, 3KL0, 3KL3 and 3KL5 for XynC, and 2Y24 for XynA) and in surface representation (D, E and F, respectively); CtXyn30A (G), XynC (H) and XynA (I) catalytic site with the various ligands observed and the side chains of the residues involved in ligand binding represented in sticks.
Comparison of the CtXyn30A catalytic site with several observed ligands superimposed on XynC shows that the HEPES molecule observed in the CtXyn30A catalytic site (PDB, id 4UQB) is positioned similarly to β-D-xylopyranose C (PDB molecule id , 3KL5) catalytic site. Thus, the ligand interactions involve similar residues in CtXyn30A and XynA, despite the different nature of the ligands. The A cleft is formed by the loops connecting the inner and outer β-sheets of a protein while the B cleft is located on the concave surface of the β-sandwich fold (Henshaw et al., 2004).
The temperature of the cuvette block was raised at 3°C/min and data were collected after each 3°C temperature increase.
Conclusions
Structural, biochemical and in silico determinants of ligand
Materials and Methods
- Reagents, chemicals and substrates
- Cloning, expression and purification of WT CtCBM6
- Ligand binding analysis of CtCBM6
- CtCBM6 on native-PAGE with soluble ligands
- Preparation of native-PAGE running buffer
- Preparation of protein sample loading buffer
Affinity gel electrophoresis (AGE) of CtCBM6 using Native-PAGE was performed against soluble ligands (polysaccharides) according to the method described elsewhere (Takeo, 1995; Tomme et al., 2000). Affinity gel electrophoresis (AE) using Native-PAGE was performed to determine the equilibrium association constant (Ka) of CtCBM6 against soluble polysaccharides according to the previously described method (Takeo, 1995; Tomme et al., 2000). After staining the gel with Coomassie Brilliant Blue, the migration of CtCBM6 was measured directly on the gel from the edge of the loading well to the center of the stained protein spot and the distances were assigned as migration without polysaccharide (R0, mm), migration in the presence of.
The effect of Ca2+ ions on the ligand binding affinity of CtCBM6 was also studied by incorporating 5 mM Ca2+ ions and polysaccharide into the gel before its polymerization. Qualitative analysis of CtCBM6 binding affinity to insoluble polysaccharides was performed as described previously (Boraston et al., 2000). Quantitative assessment of binding affinity using the adsorption isotherm was performed according to the method of Gilkes et al. (Gilkes et al., 1992).
The reaction was performed in a 200 µl volume containing various concentrations (1µM–25µM) of CtCBM6 and 1 mg/ml insoluble polysaccharide using the same reaction conditions as described above.
