Xylanases are xylan-degrading enzymes, belong to glycosyl hydrolases (GH). Xylanases from the two major families 10 (GH10) and 11 (GH11) catalyze the hydrolysis of internal β-, bonds of xylan backbone. Xylan is the second most abundant polysaccharide in nature. Nearly one third of the dry weight of the higher plants is xylan and therefore, xylanases have an important role in biomass conversions. Currently, the most effective application of xylanases is in prebleaching of kraft pulp to minimize the use of environmentally hazardous chemicals in the subsequent treatment stages. In recent years, therefore, attention is focused on to isolate and/or engineer the xylanases for the industrial requirements. The desirable roperties of xylanases in paper industry are stability and activity at high temperatures and alkaline pH. While he factors responsible for the thermal stability of GH10 xylanases have been analyzed, factors governing the alkaline stability of GH10 xylanases remain poorly understood.
The present thesis reports the crystal structures of an alkali thermostable GH10 extracellular endo-xylanase (BSX) from an alkalophilic organism, Bacillus sp. NG-27 in free and xylosaccharides-bound form. The enzyme was purified from the native organism and crystallized. The structure was solved by molecular replacement method. The 2.2 Å crystal structure of the native BSX enzyme is the first structure of an alkali thermostable GH10 family xylanase from an alkalophilic organism. It has unveiled unique protein properties that can form the basis for improving the thermal, alkaline stability and activity by site directed mutagenesis. The comparative study, especially in relation to GH10 xylanases, deciphered important structural features which are likely to be responsible for the alkaline stability of the enzyme. The work exemplifies the mechanism of adaptation of enzymes to function under polyextreme conditions through changes in the nature and composition of solvent-exposed residues. As apparent from the comparative study, the enhanced stability of the protein can be attributed to the surface rich in acidic residues and less number of solvent-exposed Asn as seen in BSX. This situation which may be roughly described as “acidic residues outside and Asn inside”, is a notable feature of alkali-stable GH10 xylanases from alkalophilic organisms.
In addition, the candidate has carried out the comprehensive database analysis of the occurrence of C-H…O hydrogen bonds in helices and helix termini of globular proteins. The study provides a compelling evidence that the main-chain Cα and the side-chains CH which participate in C-H…O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N-H…O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins.
Chapter 1 starts with a brief introduction of xylanases, their classifications and overall folds. At present, a little more than a dozen of crystal structures of GH10 xylanases are known and described in the literature. A brief mention about these structures and their optimum pH and temperature is outlined under a separate section. In view of the industrial importance of the study enzyme, the potential industrial and biotechnological applications of xylanases are detailed in this Chapter. A section is dedicated to describe the present study enzyme BSX, an alkali thermostable endo-xylanase from an alkalophilic bacterium, Bacillus sp. NG-27. BSX has a molecular mass of ~41 kDa and is optimally active at 343 K and at a pH of 8.4. The alkaline thermostability of the wild type BSX is likely to be industrially important. At the end, the scope of the present work is detailed.
Chapter 2 presents the purification of xylanase (BSX) from Bacillus sp. NG-27, the crystallization of the native and xylosaccharides-bound BSX, the X-ray diffraction data collection on these crystals and processing of the data. Repeated attempts to crystallize the protein expressed in the chloroplast of transgenic tobacco plant were unsuccessful. However, crystallization was achieved with the protein sample purified from the native source by hanging drop vapour diffusion method. Crystals were grown at both acidic (4.6) and basic pH (8.5). The corresponding crystallization conditions are 0.2 M MgCl2, 0.1 M sodium acetate pH 4.6 and 20% PEG 550 MME and 0.1 M aCl, 0.01 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000. Crystals grown at acidic pH were not suitable for X-ray diffraction study. Subsequently, crystal obtained at a basic pH of 8.5 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). Native BSX crystals belong to monoclinic space group C2 with unit cell parameters a = 174.5 Å, b = 54.7 Å, c = 131.5 Å and β = 131.2°. Crystals of xylosaccharides-bound enzyme were grown in a slightly modified crystallization condition of native, 0.1 M NaCl, 0.2 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000 and the enzyme was incubated with xylan prior to setting up the crystallization. Crystals belong to primitive orthorhombic space group P212121 with unit cell parameters a = 59.2 Å, b = 83.8 Å and c = 174.4 Å. A data set was collected using synchrotron radiation of wave length 1.0 Å from a cryo-cooled crystal at Spring-8 BL26B1 beam line, Japan. The Matthews coefficient VM for native and xylosaccharides- bound crystals was calculated to be 2.8 and 2.7 Å3 Da-1, respectively, suggesting two molecules in each crystal asymmetric unit. No twinning was detected in both the datasets and the overall quality of the data sets was found to be good.
Chapter 3 details the application of molecular replacement method to the structure solution of native and xylosaccharides-bound BSX, the course of iterative model building and the refinement carried out, and the quality of the final protein structure models. The native-enzyme structure solution was obtained by the molecular replacement method using as a search model the crystal structure (PDB code 1hiz) of the closest homologous, extracellular xylanase (GSX) from Geobacillus stearothermophilus. No non- crystallographic symmetry (NCS) restraint was applied between the two independent molecules in the crystal asymmetric unit at the final round of refinement. The final positional refinement of native BSX converged to R factors of R = 19.4% and Rfree = 23.5% for data between 20.0 to 2.2 Å. The final native model consists of 5704 protein atoms, two Mg2+ ions and 721 solvent water molecules. The final native model was taken as the search structure for the xylosaccharides-bound BSX and a solution with a correlation coefficient of 70.7% and an R-factor of 32.1% was obtained from the molecular replacement calculation. Unlike the native structure refinement, NCS restraint was imposed at all stages of the refinement. Bound xylosaccharides were clearly visible inthe difference Fourier electron density maps. The last round of refinement gave a model with R and Rfree of 21.8% and 25.7%, respectively. The final xylosaccharides-bound model consists of 5766 protein atoms, four Mg2+ ions, 85 atoms belong to bound xylosaccharides and 523 solvent water molecules. No residues were found in the disallowed region of the Ramachandran (φ, ψ) map for both the structures.
Chapter 4 describes the native and xylosaccharides-bound BSX crystal structures and the structural comparison of BSX with other GH10 family xylanase crystal structures for which the optimum temperature and pH are known in the literature. BSX folds as the ubiquitous (β/α)8-barrel, a common structural superfold characteristic of GH10 xylanases. The two active site glutamic acid residues, Glu149 and Glu259, are located on opposite sides of the active site cleft and their side-chains are at a distance of 5.5 Å apart suggesting the enzymatic reaction takes place by the retaining mechanism. From the structural superposition of other xylotriose-bound xylanase structures on to the xylosaccharides-bound BSX, structural plasticity in the xylotriose binding can be inferred, implying that the xylose recognition at the subsite -3 displays plasticity and is less specific as opposed to that at -1 and -2 subsites. The stacking interaction of one of the xylose moieties of the xylobiose with the Trp235 seen in BSX provides, for the first time, a structural evidence for the direct involvement of Trp235 in xylosaccharides binding.
The crystal structure revealed a metal binding site, found at the C-terminal end of catalytic domain. The presence of metal binding site was not anticipated from earlier theoretically modeled structure and biochemical studies. Further, we have shown experimentally the requirement of Mg2+ ion for the enzyme activity. We havedescribed a novel WP sequence-structure-interaction motif which is present in the (+) side of the active site region and presumably helps in the efficient binding of the carbohydrate moiety of the xylan in the active site cleft of BSX.
The structural comparison of BSX with other GH10 xylanases solved to date and characterized to be active at a pH close to neutral was done for the first time. The comparative study revealed the essential structural features which may responsible for the alkaline stability of GH10 xylanases.Briefly, the alkalophilic GH10 xylanases from alkalophilic organisms have surface abundant in acidic residues, the heat and alkaline susceptible residue Asn depleted on the protein surface and increased number of salt bridges.
Our study has unveiled the role of the nature and composition of protein surface amino acids in the adaptation of enzymes to polyextreme conditions. The observations reported in the thesis provide important lessons for engineering alkaline stability in xylanases for industrial applications and in general for the understanding of alkaline stability in related proteins.
A comparison of the surface features of the BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in polyextremophilicity of proteins, where understanding structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions.
Chapter 5 summaries the important findings of the present study from the crystal structural analysis of BSX and its comparison with non-alkalophilic GH10 xylanases. Separate sections are made on conclusions and future prospects for the study on BSX.
Chapter 6 describes the comprehensive database analysis of C-H…O hydrogen bond in helices of globular proteins. The C-H…O hydrogen bonds found in helices are predominantly of type 5 → 1 or 4 → 1.Our analysis reveals that the Cγ and Cβ hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic β-branched residue Ile to participate in 5 → 1 C- H…O hydrogen bonds involving methylene Cγ1 atom as donor in α-helices. In addition, C-H…O hydrogen bonds are present along with helix stabilizing salt bridges and to some extent compensate for the side-chain conformational entropy loss. Our analysis highlights that a multitude of local C-H…O hydrogen bondsformed by a variety of amino acid side-chains and Cα hydrogen atoms occur in helices and more so at the helix termini.
A majority of the helix favouring residues, Met, Glu, Arg, Lys, Leu and Gln which also have large side-chains with more donatable CH groups, have significant propensity to form side-chain to main-chain C-H…O hydrogen bonds in helix. The large side-chains are marked by their ability to shield from the solvent the polar atoms of the peptide backbone and at the same time participate in weak cohesive C-H…O interactions in the helix. This chapter also details the identification for the first time a novel chain reversal motif stabilized by 1 → 5 Cα-H…O interactions. The importance of these hydrogen bonds with respect to helix stability is discussed in the final section of the chapter.
Appendix A details the crystallographic and structural analyses oftwares used for the present thesis work.
Appendix B describes, in addition to the crystal structure analysis of BSX, the work carried out by the candidate on a comparative study of a thermostable xylanase from Thermoascus aurantiacus, solved in our laboratory at atomic 1.11 Å (293 K) and ultrahigh 0.89 Å (100 K) resolutions. From the comparison, we have for the first time pointed out the possibility of plasticity of ion pairs in proteins with water molecules mediating some of the alternate arrangements. The αβ-loops are relatively less flexible than the βα-loops. The β-strands are least affected structurally with the increase in temperature. Thus the TIM barrel fold in the study enzyme, though having a single domain, may be dissected into parts based on the relative flexibility and described as having a rigid core constituted by the β-barrel and a less rigid exterior formed by the surrounding α-helices.
Appendix C presents the crystallization and the preliminary X-ray characterization work done by the author of the thesis on an alkali thermostable cellulase enzyme from Thermomonospora sp. The protein is an extracellular enzyme with molecular mass of 14.2 kDa and interestingly, has the dual activity for both cellulose and xylan. The primary structure of the enzyme is not known. The enzyme was purified from the source organism and crystallized. A complete diffraction data set was collected and processed to 2.3 Å in an orthorhombic space group P212121.
Appendix D contains tables which give details about the analysed 5 → 1 Cα- H…O hydrogen bonds in helices and a novel chain reversal motif with 1 → 5 Cα-H…O hydrogen bonds.
Appendix E encloses reprints of publications which have resulted from the work reported in the thesis.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/328 |
| Date | 06 1900 |
| Creators | Manikandan, K |
| Contributors | Ramakumar, S |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Rights | I grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. |
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