Gotowa bibliografia na temat „Glycosyl Hydrolase Family 10 (GH10) Xylanase”

ABSTRACTEfficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed inEscherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.

Xylanase inhibitors inhibit the activities of microbial xylanases and seriously compromise the efficacy of microbial xylanases added to modify cereals. Cereal endogenous xylanases are unaffected by these xylanase inhibitors, but little information is available regarding their effects in improving cereal quality, a neglected potential application. As a strategy for circumventing the negative effects of xylanase inhibitors, the objective of this study was to use genetic engineering to obtain sufficient amounts of active endo-1,4-β-D-xylanase from wheat to analyze the characteristics of its structure. The endo-1,4-β-D-xylanase from wheat was heterologously expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, MALDI-TOF/TOF (MS) analyses, and enzyme activity determination confirmed 2 active endo-1,4-β-D-xylanases (EXY3 and EXY4) were successfully obtained. The molecular weights (MW) and isoelectric point (pI) of EXY3 were 36.108 kDa and 5.491, while those of the EXY4 protein were 41.933 kDa and 5.726. They both contained the same catalytic domain of GH10 xylanases from G266 to V276 and have the same catalytic site, Glu273. They shared the same putative N-glycosylation sites (N62-T63-S64 and N280–V281–S282) and 3 putative O-glycosylation sites (Ser8, Ser9, and Thr21), but EXY4 had an additional O-glycosylation site (Thr358). EXY3 was smaller than EXY4 by 51 amino acids because of a nonsense mutation and premature termination. They both had the 8-fold beta/alpha-barrel (TIM-barrel) fold. The specific activities of EXY3 and EXY4 were 152.0891 and 67.2928 U/mg, respectively. This work demonstrates a promising way to obtain wheat xylanases by genetic engineering; the properties of the enzymes indicate their potential application in cereal-based industries.

Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-β-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-β-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-β-1,4-xylanase.

Abstract The rice (Oryza sativa) genome encodes 37 putative β-1,4-xylanase proteins, but none of them has been characterized at the genetic level. In this work, we report the isolation of slim stem (ss) mutants with pleiotropic defects, including dwarfism, leaf tip necrosis, and withered and rolled leaves under strong sunlight. Map-based cloning of the ss1 mutant identified the candidate gene as OsXyn1 (LOC_03g47010), which encodes a xylanase-like protein belonging to the glycoside hydrolase 10 (GH10) family. OsXyn1 was found to be widely expressed, especially in young tissues. Subcellular localization analysis showed that OsXyn1 encodes a membrane-associated protein. Physiological analysis of ss1 and the allelic ss2 mutant revealed that water uptake was partially compromised in these mutants. Consistently, the plant cell wall of the mutants exhibited middle lamella abnormalities or deficiencies. Immunogold assays revealed an unconfined distribution of xylan in the mutant cell walls, which may have contributed to a slower rate of plant cell wall biosynthesis and delayed plant growth. Additionally, water deficiency caused abscisic acid accumulation and triggered drought responses in the mutants. The findings that OsXyn1 is involved in plant cell wall deposition and the regulation of plant growth and development help to shed light on the functions of the rice GH10 family.

ABSTRACTThe ascomyceteTrichoderma reeseiis a paradigm for the regulation and production of plant cell wall-degrading enzymes, including xylanases. Four xylanases, including XYN1 and XYN2 of glycosyl hydrolase family 11 (GH11), the GH10 XYN3, and the GH30 XYN4, were already described. By genome mining, we identified a fifth xylanase, XYN5, belonging to GH11. Transcriptional analysis reveals that the expression of all xylanases butxyn3is induced byd-xylose, dependent on the cellulase and xylanase regulator XYR1 and negatively regulated by the carbon catabolite repressor CRE1. Impairment ofd-xylose catabolism at thed-xylose reductase and xylitol dehydrogenase step strongly enhanced induction byd-xylose. Knockout of thel-xylulose reductase-encoding genelxr3, which connects thed-xylose andl-arabinose catabolic pathways, had no effect on xylanase induction. Besides the induction byd-xylose, theT. reeseixylanases were also induced byl-arabinose, and this induction was also enhanced in knockout mutants inl-arabinose reductase (xyl1),l-arabitol dehydrogenase (lad1), andl-xylulose reductase (lxr3). Induction byl-arabinose was also XYR1 dependent. Analysis of intracellular polyols revealed accumulation of xylitol in all strains only during incubation withd-xylose and accumulation ofl-arabitol only during incubation withl-arabinose. Induction byl-arabinose could be further stimulated by addition ofd-xylose. We conclude that the expression of theT. reeseixylanases can be induced by bothd-xylose andl-arabinose, but independently of each other and by using different inducing metabolites.

Endo-1,4-beta-xylanases (xylanases) are classified into 9 glycoside hydrolase families, GH5, 8, 10, 11, 30, 43, 51, 98, and 141 based on the CAZy database. The probe sequences representing the enzymes were constructed from published sequences of actual experimental studies with xylan decomposition activity. From online databases, we found one sequence belonging to the GH5 family, 6 sequences belonging to the GH8 family and 5 sequences belonging to the GH30 family exhibiting xylanase activity. Thus specific probes for xylanase GH8 and GH30 families were designed with the length of 351 and 425 amino acids respectively. The reference values for the probe of the GH8 family were defined as the sequences with maximum score greater than 168, the lowest coverage was 84%, the lowest similarity was 36%; for the probe GH30, the maximum score was greater than 316, the coverage was greater than 98%, the similarity was greater than 41%. Using the built probes, including the probe of the two GH10 and GH11 families, we found 41 xylanase-encoding sequences from the metagenomic DNA data of bacteria in Vietnamese goats’rumen. Of the 41 exploited sequences, 19 were identical to the BGI company's annotation result based on KEGG database, whereas there were 16 sequences that are not annotated by the BGI company. Total 28 of 41 exploited sequences were complete open reading frames, of which the predicted ternary structure was highly similar to the published structures of xylanase.

Glycoside hydrolase family 10 (GH10) xylanases are responsible for enzymatic cleavage of the internal glycosidic linkages of the xylan backbone, to generate xylooligosaccharides (XOS) and xyloses. The topologies of active-site cleft determine the substrate preferences and product profiles of xylanases. In this study, positional bindings and substrate interactions of TmxB, one of the most thermostable xylanases characterized from Thermotoga maritima to date, was investigated by docking simulations. XOS with backbone lengths of two to five (X2–X5) were docked into the active-site cleft of TmxB by AutoDock The modeled complex structures provided a series of snapshots of the interactions between XOS and TmxB. Changes in binding energy with the length of the XOS backbone indicated the existence of four effective subsites in TmxB. The interaction patterns at subsites −2 to +1 in TmxB were conserved among GH10 xylanases whereas those at distal aglycone subsite +2, consisting of the hydrogen bond network, was unique for TmxB. This work helps in obtaining an in-depth understanding of the substrate-binding property of TmxB and provides a basis for rational design of mutants with desired product profiles.

A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2ΔCBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2ΔCBM had an identical optimum temperature at 70 °C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2ΔCBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s−1, but this was reduced by 40% for XynRA2ΔCBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance.

Wheat (Triticum aestivum L.) is an important staple crop. Rhizoctonia cerealis is the causal agent of diseases that are devastating to cereal crops, including wheat. Xylanases play an important role in pathogenic infection, but little is known about xylanases in R. cerealis. Herein, we identified nine xylanase-encoding genes from the R. cerealis genome, named RcXYN1–RcXYN9, examined their expression patterns, and investigated the pathogenicity role of RcXYN1. RcXYN1–RcXYN9 proteins contain two conserved glutamate residues within the active motif in the glycoside hydrolase 10 (GH10) domain. Of them, RcXYN1–RcXYN4 are predicted to be secreted proteins. RcXYN1–RcXYN9 displayed different expression patterns during the infection process of wheat, and RcXYN1, RcXYN2, RcXYN5, and RcXYN9 were expressed highly across all the tested inoculation points. Functional dissection indicated that the RcXYN1 protein was able to induce necrosis/cell-death and H2O2 generation when infiltrated into wheat and Nicotiana benthamiana leaves. Furthermore, application of RcXYN1 protein followed by R. cerealis led to significantly higher levels of the disease in wheat leaves than application of the fungus alone. These results demonstrate that RcXYN1 acts as a pathogenicity factor during R. cerealis infection in wheat. This is the first investigation of xylanase genes in R. cerealis, providing novel insights into the pathogenesis mechanisms of R. cerealis.

Xylanases (EC 3.2.1.8) are glycosyl hydrolases that catalyze the hydrolysis of internal β-1,4 glycosidic bonds of xylan backbones, and have potential economical and environment friendly applications in the paper pulp, food, animal feed, detergent industries, bio-ethanol and bio-energy production systems. A xylanase from Bacillus sp. NG-27 (BSX), which is an extracellular endoxylanase, belonging to glycosyl hydrolase family 10 (GH10), shows optimum activity at a temperature of 70 °C and at a pH 8.5. It has a (β/α)8-triosephosphate isomerase (TIM) barrel fold, which has been studied concerning its function, structural properties, design and evolution. BSX, apart from thermo-alkalophilic features, shows resistance to SDS denaturation and protease K degradation. Hence, BSX serves as an important model system for fundamental understanding of the structure-stability-evolution relations of the ubiquitous TIM barrel fold. While the factors responsible for the thermal stability of GH10 xylanases have been analyzed, the improvement of thermostability of already thermostable enzymes is an important challenge. In general, there are large differences in optimal temperature (Tm) between hyperthermostable proteins with respect to their mesophilic homologs, indicating considerable scope available for introducing novel protein engineering approaches to improve protein stability. Thermostability and thermotolerance are of particular importance for industrial enzymes, because higher operating temperatures allow higher reactivity, higher bioavailability, higher process yield, lower viscosity, and reducing the risk of contamination. Thus, finding enzymes that can function at high temperatures has immense industrial importance and constitutes an active area of research. Earlier studies on enzymatic activity and thermostability of a recombinant BSX (RBSX) with different extreme N-terminus mutants by biochemical/biophysical methods showed that a single amino acid substitution (Val1→Leu) markedly enhanced the thermostability of recombinant xylanase from 70 °C to 75 °C without compromising its catalytic activity and showed higher cooperativity in the thermal unfolding transition. Conversely, substitution of Val1→Ala (V1A) at the same position decreased the stability of the protein from 70 °C to 68 °C. Furthermore, it was observed that substitution of Phe4 by Ala decreased the stability by ~4 °C whereas substitution of Trp6→Ala and Tyr343→Ala decreased the stability by ~10 °C with respect to RBSX. On the other hand, substitution of Phe4 by another aromatic residue Trp (F4W) did not change the stability and activity of RBSX. However, structural details were not available at that time, precluding any structure-based rationalization of stability changes resulting from a single amino acid substitution. The thesis reports the crystal structures of a recombinant xylanase from Bacillus sp. NG-27 (RBSX) and its various N-terminal and C-terminal mutants namely V1A, V1L, F4A, F4W, W6A, and Y343A. The crystal structure of RBSX (PDB ID: 4QCE) was solved at a resolution of 2.35 Å whereas those of V1A mutant (PDB ID: 4QCF) and V1L mutant (PDB ID: 4QDM) were solved at a resolution of 2.26 Å and 1.99 Å respectively. On the other hand, the crystal structure of F4A was solved at a resolution of 2.23 Å whereas F4W, W6A, and Y343A mutants were solved at a resolution of 2.22 Å, 1.67 Å, and 2.30 Å respectively. The availability of experimentally determined RBSX structure and its various mutant structures has enabled a critical examination including from a network perspective, of factors influencing thermal stability. The crystal structures in combination with computational analysis have provided valuable insights into the structural features that govern protein thermostability. The thesis candidate established a link between N-terminal to C-terminal contacts and RBSX thermostability. The study reveals that augmenting N-terminal to C-terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. Perhaps, for the first time, the study provides a network perspective of N-terminal to C-terminal interactions and shows that the stabilizing interactions are not restricted to terminal regions but propagate to different parts of the protein structure. Furthermore, analysis of structures of different aromatic mutants of RBSX and structural bioinformatics studies were combined to understand the role of long-range aromatic cluster in the form of 'aromatic-clique' in the thermal stabilization of proteins. The results highlight an additional source of stability in thermophilic proteins, which could arise due to the prevalence of aromatic-cliques. In addition, the work exemplifies the experimental evidence specifically through long-range aromatic clique, in reiterating the role of interactions between N- and C-termini in protein stabilization. The thesis candidate demonstrated the experimental evidence depicting the role of partially solvent exposed tryptophan residues in shielding a surface pocket, which influenced the solvation of backbone atoms and stability of the RBSX enzyme. The candidate carried out a comprehensive database analysis of available crystal structures to look into the possible role of partially exposed tryptophan in hyperthermophilic proteins. The study provides strong evidence that partially exposed tryptophan side-chain is recruited in hyperthermophilic proteins for occluding potential surface pockets, to provide backbone solvent shielding and local stabilization. The overall structure of this thesis is further explained through a chapter wise description below: Chapter 1 | An introduction and outline of the thesis This chapter starts with a general introduction about the diversity of microorganisms and their ability to thrive in extreme environments such as high temperature. The research on these enterprising organisms offers not just the insights into the resilience of life on earth or possibilities of life elsewhere in the universe but also can provide exciting opportunities for a variety of industrial, environmental, biomedical, and pharmaceutical applications. While the adaptation of the cell inventory is important, it is a challenge for proteins to overcome high temperature in order to remain folded in the correct three-dimensional structure while maintaining adequate flexibility for their desired function. Hence, elucidation of the molecular basis of protein stability at extreme temperature continues to attract researcher over a board range of disciplines. The various structural features responsible for protein stability are outlined and the basic structural and molecular strategies for the adaptation to high temperatures revealed by structure analysis are delineated. Of all potentially deactivating factors of protein stability, temperature is the best studied. A brief outline of the strategies and approaches for the design of proteins to meet the desirable properties such as increased thermal stability are presented whereas the structural features responsible for stability of triosephosphate isomerase (TIM)-barrel fold is outlined under a separate section. Subsequently a short introduction of family 10 (GH10) xylanases, which has the ubiquitous TIM-barrel fold and their classifications are presented. A section is dedicated to describe various thermostable GH10 xylanases, their structural features responsible for stability, and current and potential biotechnological applications. At the end, the scope of the present work is detailed. Chapter 2| Crystallization, Data Collection and Data processing of recombinant BSX (RBSX) and its different variants: Chapter 2 presents the purification of recombinant xylanase from Bacillus sp. NG-27 (RBSX), its N-terminal variants (V1A, V1L), and aromatic variants (F4W, F4A, W6A, and Y343A). The expression and purification of RBSX and its variants were carried out at the laboratory of our collaborator Prof. V. S. Reddy, Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. Initial crystallization trials were screened by hanging drop vapor diffusion method and micro-batch diffusion method using crystallization-screening kits (Crystal Screen and Crystal Screen 2) from Hampton Research, USA and a laboratory made screen, which was based on reported crystallization condition of native BSX. After a few rounds of trials and optimization of the crystallization condition, diffraction quality rod shaped crystals of recombinant BSX (RBSX) were obtained within ten days, when 2-µl protein solution (10 g/ml) was mixed with 2-µl reservoir solution composed of 0.12 M MgCl2, 0.1 M NaCl, 0.1M Tris-HCl pH 8.5 and 15% PEG 8000. Subsequently, crystal was used for X-ray data collection and it diffracted X-rays to better than 2.2 Å at the home source at cryo-temperature (100 K). RBSX crystals belong to orthorhombic space group P212121 with unit cell parameters a = 54.77 Å, b = 75.65 Å, c = 179.91 Å and α = β = γ =90°. A three-dimensional screening grid was prepared based on crystallization condition of RBSX by carefully varying salt concentration (NaCl and MgCl2 from 10mM to 300mM in the interval of 10mM), different PEG variants (PEG 1000, PEG 3350, PEG 4000, PEG 8000, and PEG 10000) in the range of 5% to 20%. Tris-HCl buffer of pH 8.0 and of pH 8.5 was used in the concentration range of 0.05M and 0.1M respectively. Rod shaped crystals were obtained using hanging drop vapor diffusion method from the condition of 0.1M NaCl, 80mM MgCl2, 0.05M Tris-HCl pH 8.5 and 18 % PEG 8000 and 0.1M NaCl, 60mM MgCl2, 0.1M Tris-HCl pH 8.5 and 16 % PEG 8000 for V1L mutant and V1A mutant respectively. The diffracting crystals of F4A mutant were obtained from the condition of 0.1M NaCl, 140mM MgCl2, 0.05M Tris-HCl pH 8.5 and 15% PEG 8000 by using hanging drop vapor diffusion method. On the other hand, F4W and Y343A, crystals were grown by micro-batch diffusion method containing 1.1-μ1 ratio of protein and crystallization solution of 0.1M NaCl, 120mM MgCl2, 0.1M Tris-HCl pH 8.5 and 18% PEG 8000 and 0.1M NaCl, 150mM MgCl2, 0.1M Tris-HCl pH 8.5 and 15 % PEG 6000 respectively . W6A mutant crystals were grown by hanging drop vapor diffusion method of 0.1M NaCl, 160mM MgCl2, 0.05M Tris-HCl pH 8.5 and 20% PEG 8000. All the crystals were obtained at 20 °C-22 °C in 5-10 days, and were used for diffraction experiments (details in the table below). Table 1 Protein Space a b c α β γ X-ray source PDB group (Å) (Å) (Å) (°) (°) (°) ID RBSX P212121 54.77 75.65 176.91 90 90 90 Home-source 4QCE V1A C2 73.57 80.12 69.90 90 110.81 90 Home-source 4QCF V1L P212121 54.88 76.58 176.73 90 90 90 Synchrotron 4QDM F4W P212121 55.27 77.32 176.75 90 90 90 Home- source 5EB8 F4A P212121 52.62 67.71 181.54 90 90 90 Home- source 5EFF W6A P212121 54.99 76.60 181.54 90 90 90 Synchrotron 5EFD Y343A C2 73.86 80.11 69.21 90 111.19 90 Home- source 5EBA The quality of all dataset was assessed by SFCHECK. The data sets were found appropriate and useful for structure determination as discussed in Chapter 3. Chapter 3 | Molecular Replacement, Model Building, Refinement, validation of recombinant xylanase (RBSX), and different mutant structures: Chapter 3 details the application of molecular replacement method to the structure solution of RBSX structure, N-terminal and aromatic mutants of RBSX, the course of iterative model building and the refinement carried out and the quality of the final protein structure models. The structure solution for all the structures was obtained by the molecular replacement (MR) method with the program PHASER-MR in the PHENIX package using a search model of native-enzyme (2F8Q). The asymmetric unit of RBSX, V1L, F4A, F4W, W6A crystals was expected to contain two molecules whereas V1A and Y343A crystal was expected to contain one molecule as indicated by Matthews’s coefficient calculation. The final round of refinement was carried out with restrained refinement with TLS parameters for all the structures. The most essential refinement statistics of the final model of RBSX, V1A, and V1L mutant structures are given in Table 2 whereas the same for aromatic mutant structures, F4A, F4W, W6A, and Y343A are given in Table 3. Table 2 Refinement Statistics RBSX V1A V1L Resolution (Å) 27.7-2.32 26.8-2.26 40.2-1.96 Rwork / Rfree (%) 17.9/22.7 17.4/22.5 15.2/19.0 Average B-factors (Å2) Protein 21.6 26.3 13.9 Ligand/ion 15.6 26.4 18.74 Water 20.6 27.2 23.2 RMSD Bond distance (Å) 0.007 0.005 0.019 Bond angles (◦) 1.123 0.955 1.802 Luzzati coordinate 0.279 0.269 0.175 error (Å) Working set Table 3 Refinement Statistics F4A F4W W6A Y343A Resolution (Å) 18.15-2.23 18.97-2.22 32.1-1.67 34.03-2.30 Rwork / Rfree (%) 17.8/24.0 16.8/21.0 15.68/18.58 17.8/23.0 Average B-factors (Å2) Protein 13.1 19.5 16.7 26.8 Ligand/ion 14.2 20.0 21.4 26.1 Water 11.5 28.9 25.9 30.7 RMSD Bond distance (Å) 0.0144 0.0088 0.0139 0.0063 Bond angles (◦) 1.593 1.228 1.5995 1.0951 Luzzati coordinate 0.261 0.252 0.176 0.293 error (Å) Working set Chapter 4 | Mutations at the extreme N-terminus modulate thermostability of RBSX: Implications of interactions between termini for stability This chapter details the structural analysis of RBSX and its various extreme N-terminus mutations in relation to their different thermostability scale. Although several factors have been attributed to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a RBSX, which has the ubiquitous (β/α)8-TIM (Triosephosphate isomerase) barrel fold showed that just a single mutation, Valine1→Leucine (V1L), though not part of any secondary structural element, markedly enhanced the stability from 70 °C to 75 °C without loss of catalytic activity. Conversely, substitution of Valine1→Alanine (V1A) at the same position decreased the stability of the enzyme from 70 °C to 68 °C. To gain structural insights as to how a single extreme N-terminus mutation can markedly influence the thermostability of the enzyme, the candidate has determined the crystal structure of RBSX and two mutants. Based on computational analysis of their crystal structures including residue interaction network, a link was established between N- to C-terminal contacts and RBSX thermostability. The study reveals that augmenting N- to C-terminal non-covalent interactions is associated with the enhancement of the stability of the enzyme. Perhaps, for the first time, the study provides a network perspective of N-terminal to C-terminal interactions and shows that the stabilizing interactions are not restricted to terminal regions but propagate to different parts of the protein structure. In addition, several lines of evidence were discussed that point to support the structural coupling between the chain termini and implications of stability changes in different proteins. It is proposed that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising on enzymatic activity, or in general, protein function, in diverse folds where N- and C-termini are in close proximity. Chapter 5 | Role of long-range aromatic cluster in the structural stability of RBSX Chapter 5 describes the different aromatic mutant crystal structures of RBSX namely F4W, F4A, W6A, and Y343A and the structural comparison with the RBSX crystal structure. Systematic studies of different alanine mutations (F4A, W6A, and Y343A) to disrupt this aromatic cluster showed that substitution of Phe4, Trp6, and Y343 by alanine drastically decreased the stability of recombinant BSX (RBSX). It was observed that substitution of Phe4 by Ala (F4A) decreased the RBSX stability by ~5 °C whereas substitutions of Trp6 by Ala (W6A) and Tyr343 by Ala (Y343A) markedly decreased the stability of the enzyme by ~10 °C. On the other hand, substitution of Phe4 by Trp (F4W) did not result any change in its thermal unfolding pattern of the enzyme. We observed that the mutated amino acid residues (Phe4, Trp6, and Tyr343) in the RBSX structure are part of an ‘aromatic-clique’. An aromatic-clique is defined as a cluster of aromatic residues in which each residue interacts with all other residues within the cluster through aromatic interactions. The study reveals that the decreased stability shown by F4A, W6A, and Y343A mutants resulted from cumulative effects in the loss of aromatic interactions and disruption of aromatic-clique, and reduced van der Waal interactions. In addition, the work exemplifies the importance of interactions between N-terminal and C-terminal through aromatic contacts or packing in folding and stability of the TIM-barrel fold protein. The structure based multiple sequence alignment of RBSX with other GH10 xylanase from Bacillus organisms revealed that aromatic-clique of interest is fully conserved in B. halodurans (BHX) and Bacillus firmus (BFX) xylanases, which are thermostable in nature, like RBSX. On the other hand, this aromatic-clique is not conserved in the GH10 xylanases from Bacillus N137, Bacillus alcalophilus, which are reported as thermo-labile in nature. Furthermore, analysis of available crystal structures of different thermostable xylanases from GH10 family showed the prevalence of aromatic-clique that may be playing a critical role in their structure-stability and folding. Lastly, a comprehensive analysis of homologous pairs of proteins from (hyper)thermophilic and mesophilic organisms was carried out and observed the high occurrence of aromatic-cliques in the thermophilic proteins in comparison to their mesophilic homologs. These results highlight an additional source of stability in thermophilic proteins, which can arise due to the prevalence of aromatic-cliques. The findings reported in the thesis provide important lessons for engineering xylanases for industrial applications. The strategy of mutations based on clustering of aromatic pairs in the form of ‘aromatic-clique’ may be effectively applied to other enzymes and provides new insights for engineers to design proteins for biotechnological applications. Chapter 6 | Tryptophan occludes surface pocket: Implications for protein stability Chapter 6 describes the structural feature of a partially exposed tryptophan residue, which effectively occludes a surface pocket and plays a critical role in RBSX thermo-stabilization. As a part our long-standing interest in the structural analysis of thermostable proteins, it was observed that just a single mutation, W6A of a recombinant xylanase (RBSX) from Bacillus sp. NG-27 decreased the stability from 70 °C to 60 °C. To gain structural insights into how a single mutation W6A can remarkably influence the thermostability of the enzyme, we determined the crystal structure of W6A mutant and compared the same with the crystal structure of RBSX. We serendipitously observed that substitution of Trp6 by alanine (W6A) in the protein results a small surface pocket, which was shielded by the bulky side-chain of Trp6 in the native structure. Inspection of the molecular structure of native protein structure revealed that side chain of Trp6 occludes the surface pocket, sterically impeding entry of solvent molecules including water. We demonstrated the experimental evidence depicting how a partially exposed tryptophan, which was shielding a surface pocket (tryptophan-shield), can directly influence the backbone solvation, and modulate the stability of the enzyme. Furthermore, computational analysis of high-resolution structures of hyperthermophilic proteins reveals that bulky and aromatic indole side-chain of tryptophan effectively occludes surface pockets in several hyperthermophilic proteins. The study provides a strong evidence that partially exposed tryptophan side-chain is recruited in hyperthermophilic proteins for occluding potential surface pockets to provide backbone solvent shielding and local stabilization. Chapter 7 | Summary and future direction Chapter 7 summaries the important findings of the present study from the crystal structure and computational analysis of a recombinant xylanase (RBSX) and its various N-terminal and C-terminal mutants and also outlines the future direction of the work. Appendix A details SFCHECK output for the processed data for all the structures reported in the thesis. Appendix B Reprints of the publications