Glycoside hydrolases from Rhodothermus marinus Modular organisation and structure-function relationships

University dissertation from Maher Abou Hachem, Kämnärsvägen 13, E105, 226 46 Lund, Sweden

Abstract: The thermophilic bacterium Rhodothermus marinus 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 Escherichia coli. 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.

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