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

Abou-Hachem, Maher LU (2003)
Abstract
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Professor Liebl, Wolfgang, Georg-August-Universität Göttingen, Germany
organization
publishing date
type
Thesis
publication status
published
subject
keywords
calcium-binding, modular, endoglucanase, thermostable, CBM, xylanase, Biotechnology, Bioteknik
pages
160 pages
publisher
Maher Abou Hachem, Kämnärsvägen 13, E105, 226 46 Lund, Sweden,
defense location
lecture hall B, Center for Chemistry and Chemical Engineering, Sölvegatan 39, Lund
defense date
2003-03-28 13:15
external identifiers
  • Other:ISRN: LUTKDH/TKBT--03/1065--SE
ISBN
91-7422-014-4
language
English
LU publication?
yes
id
2bbc271d-1bc4-4dca-839e-214031c8aa65 (old id 465573)
date added to LUP
2007-10-14 13:47:18
date last changed
2016-09-19 08:45:15
@phdthesis{2bbc271d-1bc4-4dca-839e-214031c8aa65,
  abstract     = {The thermophilic bacterium &lt;i&gt;Rhodothermus marinus&lt;/i&gt; 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 &lt;i&gt;Escherichia coli&lt;/i&gt;. 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.},
  author       = {Abou-Hachem, Maher},
  isbn         = {91-7422-014-4},
  keyword      = {calcium-binding,modular,endoglucanase,thermostable,CBM,xylanase,Biotechnology,Bioteknik},
  language     = {eng},
  pages        = {160},
  publisher    = {Maher Abou Hachem, Kämnärsvägen 13, E105, 226 46 Lund, Sweden,},
  school       = {Lund University},
  title        = {Glycoside hydrolases from Rhodothermus marinus Modular organisation and structure-function relationships},
  year         = {2003},
}