The heteroxylan backbone consists of β-(1→4)-linked xylose residues, which is often substituted with methylated α-D-glucopyranosyl uronic acid and α-L-arabinofuranosyl, ferulic acid, p-coumaric acid in the side chain (Ordaz- Ortiz and Saulnier, 2005). The complex structure of xylan requires the synergistic action of a set of enzymes such as endo-β-xylanase, α-glucuronidase, β-xylosidase, α-arabinofuranosidase, p-coumaric acid esterase, acetyl xylan esterase and ferulic acid esterase for its complete hydrolysis (Sharma et al ., 2018). Analysis of the carbohydrate composition and structural characterization of the extracted xylan showed that the main chain consists of a xylose backbone replaced by a 4-O-methyl glucuronic acid side chain.
General Introduction 1
Structure of lignocellulosic biomass
- Cellulose…
- Lignin
- Hemicelluloses
- Mannans
- Xyloglucans
- Arabinans
- Xylans
Lignin is mainly present in the secondary plant cell walls and constitutes one of the main components of the plant cell wall. Arabinans are widely distributed in the various plant tissues and important structural components of the cell wall.
Enzymatic degradation of lignocellulosic biomass…
- Glycoside hydrolase
- Cellulolytic enzymes
- Hemicellulolytic enzymes
Endoglucanases (EC 3.2.1.4) randomly catalyze the β-(1,4)-glycosidic bonds (internal bonds) in the amorphous regions of the polysaccharides and produce oligosaccharides. The hemicellulolytic enzymes involved in the complete conversion of heteroxylans are called xylanolytic enzymes.
Xylanolytic enzymes
- Family 43 glycoside hydrolases
- Family 30 glycoside hydrolases
- Family 10 glycoside hydrolase
- Mechanism of action of xylanolytic enzymes
- Retaining mechanism
- Inverting mechanism
- Industrial production and application of xylanolytic enzymes…
Out of these, the crystal structure of only 1 modular arabinofuranosidase (BsAXH-m2,3) from Bacillus subtilis containing a catalytic module of family GH43 and family 6 carbohydrate-binding module (CBM6) as a non-catalytic module (PDB Id: 3C7E ) is have been reported and available in the PDB (Vandermarliere et al., 2009). The GH10 family contains a total of 3851 protein sequences in the CAZy database as of September 2019 (http://www.cazy.org/GH10.html).
The microorganism
Pseudopedobacter saltans is a Gram-negative soil-associated bacterium and one of the currently known 32 species in the genus Pedobacter within the family Sphingobacteriaceae.
Significance of the investigation
However, several questions remain unanswered about the conformational dynamics, molecular arrangement and protein stability of these modular and non-modular enzymes in solution. In this regard, the present study deals with the structural organization, dynamics and functional characterization and application of modular and non-modular xylanolytic enzymes from Clostridium thermocellum and Pseudopedobacter saltans.
Objectives of the present study
- Specific objectives
The conformational dynamics, molecular arrangement and protein stability in solution will be determined by Small Angle X-ray Scattering (SAXS) analysis and computational modeling of full-length CtXynGH30. Extraction and characterization of xylan from neem sawdust will be explored in the final objective.
Characterization of the isozymes of glucuronoxylan xylanohydrolase in the presence of a native cell wall substrate. Plant cell wall degradation by saprophytic Bacillus subtilis strains: Gene clusters responsible for rhamnogalacturonan depolymerization.
Introduction
These enzymes act cooperatively in the extracellular environment to deconstruct complex carbohydrates, thereby generating simple sugars that are used by microbes as an energy source (Gilbert et al., 2008). Enzymes that are active in arabinose-linked glycosidic linkages are mainly located in the GH43, GH51, GH54, and GH62 families ( Cantarel et al., 2009 ).
Material and methods
- Material
- Sequence retrieval and analysis of modular CtAraf43
- Expression and purification of CtAbf43A
- Site-directed mutagenesis of CtAbf43A
- Enzyme activity determination
- Crystallization, data collection, structure determination and refinement. 47
- Sequence analysis of CtAraf43
- Overall structure of CtAbf43A
- Substrate recognition by CtAbf43A
- Structural determinants of specificity in GH43_16
The structure of the cubic form was solved by molecular replacement using BALBES (Long et al., 2008). The secondary structure of the five-sheet β-screw fold is colored differently for each blade. Superposition of CtAbf43A with BsAXH-m2,3 in complex with xylotetraose (Vandermarliere et al., 2009) revealed the structural properties of the different subsites of GH43_16 enzymes that modulate arabinoxylan specificity (Fig. 2.5).
Inspection of the primary sequence of GH43_16 members suggests that this conservation spans the entire subfamily. In contrast, the topology of the substrate-binding cleft is similar in the two enzymes.
Conclusion
Crystal structure of an exo-1,5-α-L-arabinofuranosidase from Streptomyces avermitilis provides insight into the mechanism of substrate discrimination between exo- and endotype enzymes in glycoside hydrolase family 43. Advances in understanding the molecular basis of plant cell wall polysaccharide recognition using carbohydrate-binding modules . Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared to other members of the same family.
Small angle X-ray scattering based structure, modelling and
Material and methods
- Sequence retrieval and analysis of modular CtAraf43
- Molecular modelling and validation of CtAraf43
- Molecular dynamic simulation of the CtAraf43 structure
- Expression and purification of CtAraf43
- Small Angle X-ray Scattering (SAXS) of CtAraf43
- SAXS data analysis
The subcellular localization of CtAraf43 was analyzed using the SignalP v4.0 server ( Petersen et al., 2011 ). Modification in the secondary structure of CtAraf43 was performed by DSSP v3.0 (Touw et al., 2014) compiled with Gromacs v5.14. CtAraf43 purification was performed using immobilized metal ion affinity chromatography (IMAC) using 5 ml Sepharose column (HisTrap Chelating, GE Healthcare, USA) as reported by Ahmed et al., (2013).
The molecular mass of CtAraf43 was estimated from its distribution pattern from the SAXS Mow web server ( Fischer et al., 2010 ). The molecular envelope of CtAraf43 was reconstructed by the ab-initio modeling method implemented in DAMMIF using the SAXS I(Q) profile and the results obtained from its pairwise distribution of interatomic vectors (Svergun et al., 2001).
Results and discussion
- Sequence analysis of CtAraf43
- Structure modelling and validation of CtAraf43
- Molecular dynamics simulation of the CtAraf43 structure
- Solution structure analysis of CtAraf43 by Small Angle X-ray
The structural topology analysis of CtAraf43 from PDBSum web server also confirmed with the modeled structure (Fig. 3.2B). The SAXS pattern of CtAraf43 at concentrations 1.2 mg/ml and 4.7 mg/ml showed no inter-particle interaction and aggregation (Fig. 3.5B). The molecular envelope of CtAraf43 at 1.2 mg/ml exhibited Miniature Dashhund shape and at 4.7 mg/ml shows 2-fold rotational symmetry like envelope.
SAXS analysis of CtAraf43 revealed its monodisperse nature at both concentrations (1.2 mg/mL and 4.7 mg/mL). SAXS analysis of CtAraf43 suggested elongated structures with monomeric conformation at 1.2 mg/mL and dimeric conformation at 4.7 mg/mL.
Determination of the molecular weight of proteins in solution by a single measurement of small-angle X-ray scattering on a relative scale. Molecular determinants of substrate specificity revealed by the structure of Clostridium thermocellum arabinofuranosidase 43A from subfamily 43 of glycosyl hydrolase family 16. Molecular organization and stability of the solute proteins of Clostridium thermocellum glucuronoxylan endo-β- 1,4 glycolase of glycosylase family 43. the family.
Molecular organization of glucuronoxylan endo-β-1,4-xylanase of
Material and methods
- Retrieval of the protein sequence of modular CtXynGH30
- Secondary structure analysis of CtXynGH30
- Molecular modeling
- Protein melting studies of CtXynGH30
- Analysis of the contribution of modules in CtXynGH30
- CtXyn30A and CtCBM6 binding assay by ITC
- CtXyn30A and CtCBM6 interaction by MALDI-TOF
- Assay of CtXyn30A:CtCBM6 mixture, CtXynGH30 and
- SAXS data analysis
The subcellular localization of CtXynGH30 was analyzed using the SecretomeP 2.0 server ( Bendtsen et al., 2005 ). The recovered amino acid sequence of CtXynGH30 (559 aa long out of 630 aa as the C-terminal dockerin was not included in this study) was subjected to PSI-BLAST search using default parameters against a Protein Data Bank (PDB). The protein melting point of CtXynGH30 was determined using 40 µg of purified CtXynGH30 in 1 ml of 20 mM sodium phosphate buffer, pH 6.0.
Multiple frames of CtXynGH30 scattering patterns were merged using the Primus package of ATSAS 2.8 suite ( Franke et al., 2017 ). The molecular weight of CtXynGH30 was calculated from the scattering pattern by SAXS Mow Web Server (Fischer et al., 2010).
Results and discussion
- Sequence analysis of CtXynGH30
- Secondary structure analysis of CtXynGH30 by Psipred and CD
- The protein melting analysis of CtXynGH30
- Role of CtCBM6 in CtXynGH30
- Solution structure analysis of CtXynGH30 by Small Angle X-ray
The presence of 1 mM Ca2+ ions shifted the melting peak of CtXynGH30 to a higher temperature of 85ºC. The comparative SAXS pattern of CtXynGH30 at different concentrations showed that concentration effects were negligible. Guinier plot analysis of CtXynGH30 showed a linear behavior in the low q region, which confirms that the protein sample preparation is in a monodisperse state.
The overall appearance of the GASBOR-generated molecular shape of CtXynGH30 matched well with its refined SREFLEX structure (Fig. 4.6 C & D). Secondary structure analysis of CtXynGH30 by CD revealed the presence of 28.25% α-helix and 40.5% β-sheet.
Conservation in the mechanism of glucuronoxylan hydrolysis revealed by the structure of glucuronoxylan xylanohydrolase (CtXyn30A) from Clostridium thermocellum. Structural analysis of the glucuronoxylan-specific Xyn30D and its attached CBM35 domain provides insights into the role of modularity in specificity. In silico structural characterization and molecular docking studies of the first glucuronoxylan-xylanohydrolase (Xyn30A) from glycosyl hydrolase family 30 (GH30) from Clostridium thermocellum.
The family 6 carbohydrate binding module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A. A new member of family 30 glycoside hydrolase subfamily 8 glucuronoxylanendo-β-1,0-xylanase (CtXynlanGH-1,0) from Clostridium thermocellum orchestrates catalysis on arabinose-decorated xylans.
SAXS and comparative modeling based structure analysis of endo-
Material and methods
- Amino acid sequence retrieval and analysis
- Comparative modeling, refinement and structure assessment of
- Secondary structure analysis of PsGH10A
- Molecular docking analysis of PsGH10A
- Protein melting analysis of PsGH10A
- Small Angle X-ray Scattering Analysis (SAXS) of PsGH10A
The molecular mass analysis of PsGH10A was determined by Protparam tool (https://web.expasy.org/protparam/). Multiple template-based comparative modeling approach was used to construct three-dimensional structure of PsGH10A. The secondary structure of PsGH10A was predicted using RaptorX property prediction server ( Wang et al., 2016 ).
The protein melting curve of PsGH10A was constructed by plotting the change in absorbance at 280 nm (A280) and at different temperatures using a double-beam UV-visible spectrophotometer with peltier (Varian, Cary 100-Bio) according to the previously described method (Ahmed et al., 2013). The molecular mass of PsGH10A was estimated by the SAXS MoW program using SAXS data (Fischer et al., 2010).
Results and discussion
- Sequence analysis of PsGH10A
- Structure modeling and validation of PsGH10A
- Energy minimization and structure validation of PsGH10A
- Secondary structure analysis of PsGH10A
- Molecular docking analysis of PsGH10A
- Protein melting analysis of PsGH10A
- Low resolution structure analysis of PsGH10A by SAXS
3-dimensional structure analysis of PsGH10A. A) 3D structure showing TIM-barrel (β/α) 8-fold, B) PsGH10A showing salad bowl shape with α-helices, β-strands and connecting loops labeled, C) Surface view of PsGH10A showing loops 4, 7, and 8 (L4, L7, and L8) forming the catalytic cleft, D) catalytic residues, Glu156 and Glu263, shown in purple, with the distance between catalytic residues shown as a dashed line, and E) topology diagram of PsGH10A, showing the N and C-terminal regions with α-helices, β-strands and connecting loops. The quality factor of the modeled PsGH10A structure with the ERRAT plot was 94.45%, which further confirmed the excellent quality of the PsGH10A model (Figure 5.4E). Far-UV circular dichroism (CD) spectrum of PsGH10A analyzed with the CONTIN program available on Dichroweb.
This indicated that the active site of PsGH10A can adapt to xylotetraose. Therefore, the analysis of the melting curve of PsGH10A was also performed in the presence of 2 mM Mn2+ or Mg2+ ions and in combination with EDTA.
Conclusion
The carbohydrate composition was determined by hydrolyzing the extracted xylan (5 mg) with 2M trifluoroacetic acid (TFA) in a boiling water bath for 3 hours (Sharma, Rajulapati & Goyal, 2019). Surface properties of extracted xylan from neem were investigated using a field emission scanning electron microscope (Carl Zeiss, Model-Gemini 300, Germany). The dried product was dissolved in 200 μl of deionized water and the xylooligosaccharides yield (total carbohydrate content) was determined using the phenol-sulphuric acid method (Dubois, Gilles, Hamilton, Rebers & Smith, 1956). The qualitative estimation and identification. Xylooligosaccharides were carried out by running 0.5 μl of concentrated sample on TLC plate using Chloroform:Acetic acid:Water in a ratio of 6:7:1 as a mobile phase.
The Carbazole test showed that the extracted xylan from neem sawdust contains ~14% glucuronic acid (139±5 µg/mg xylan). Fourier Transform Infrared (FT-IR) spectroscopic analysis of extracted xylan (A), TOCSY-HSQC NMR (B) and HMBC NMR (C) analysis of extracted xylan.
