Glycoside hydrolases from Rhodothermus marinus Modular organisation and structure-function relationships
Research output: Thesis › Doctoral Thesis (compilation)
The thermophilic bacterium <i>Rhodothermus marinus</i> produces several thermostable glycoside hydrolases. The studies presented in this thesis were performed on two enzymes, belonging to glycoside hydrolase families 10 and 12, produced by this microorganism. The family 10 xylanase, Xyn10A, is modular in architecture consisting of five domains or modules. The two isolated N-terminal modules were produced and characterised. These modules were proven to be carbohydrate-binding modules (CBMs) belonging to a novel subdivision of family 4 CBMs. Both modules display affinity for xylans, b-glucans, and to a less extent non-crystalline cellulose. The structure of the second of these binding modules (CBM4-2), solved by NMR, featured a b-sandwich with jelly roll-topology. Structural details and substrate titrations provided valuable insight on the determinants of specificity of the module. Both the Xyn10A CBMs and the third domain in the enzyme were shown to bind calcium ions, which had a pronounced effect on their thermostabilities. In addition, modular interactions seemed to enhance the stability of the enzyme, since deletion mutants were less stable than the full-length enzyme. No specific function could be ascribed the third domain of Xyn10A, while evidence suggested that the fifth domain is a novel module type that mediates cell-attachment. The primary structure of the family 12 endoglucanase Cel12A was analysed. These analyses showed that the catalytic module of this enzyme is preceded by a linker sequence and a putative signal peptide that destabilised the enzyme and impaired its expression in <i>Escherichia coli</i>. Designing mutants lacking this signal peptide readily solved the stability and production problems and these mutants retained their thermostability and activity. Finally, fusion proteins between the Xyn10A CBMs and the catalytic module of Cel12A were produced and some of their properties are reported.
|Research areas and keywords||
Subject classification (UKÄ) – MANDATORY
|Award date||2003 Mar 28|
|Publication status||Published - 2003|
Defence details Date: 2003-03-28 Time: 13:15 Place: lecture hall B, Center for Chemistry and Chemical Engineering, Sölvegatan 39, Lund External reviewer(s) Name: Liebl, Wolfgang Title: Professor Affiliation: Georg-August-Universität Göttingen, Germany --- Article: I. Abou Hachem, M., Nordberg Karlsson, E., Bartonek-Roxå, E., Raghothama, S., Simpson, P.J., Gilbert, H.J., Williamson, M.P. and Holst, O. (2000) Carbohydrate binding modules from a thermostable Rhodothermus marinus xylanase: Cloning, expression and binding studies. Biochemical Journal 345, 53-60A. Article: II. Abou Hachem, M., Nordberg Karlsson, E., Simpson, P.J., Williamson, M.P., Seller, P., Linse, S., Gilbert, H.J., Bolam D.N., and Holst, O. (2002) Calcium binding and thermodynamic studies on the thermostable carbohydrate binding modules of Xyn10A Rhodothermus marinus. Biochemistry 41, 5720-5729B. Article: III. Simpson, P.J, Jamieson, S.J., Abou-Hachem, M., Nordberg Karlsson, E., Gilbert, H.J., Holst, O., Williamson, M.P. (2002) The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase. Biochemistry 41, 5712-5719B. Article: IV. Abou Hachem, M., Olsson, F., and Nordberg Karlsson, E. Probing the stability of the modular family 10 xylanase from Rhodothermus marinus. (manuscript) Article: V. Wicher, K.B., Abou-Hachem M., Halldorsdottir, S., Thorbjarnadottir, S.H., Eggertsson, G., Hreggvidsson, G.O., Nordberg Karlsson E., Holst O. (2001) Deletion of a cytotoxic, N-terminal putative signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus. Applied Microbiology and Biotechnology 55, 578-84C. Article: VI. Abou Hachem, M., Ramchuran, S.O., Oxhamre, C., Crennell, S.J., Holst, O. Nordberg Karlsson, E. Chimeric thermostable glycoside hydrolases: Fusion of family 4 carbohydrate binding modules to a family 12 endoglucanase catalytic module (manuscript).