Probing the stability of the modular family 10 xylanase from Rhodothermus marinus.
The thermophilic bacterium Rhodothermus marinus produces a modular xylanase (Xyn10A) consisting of two N-terminal carbohydrate-binding modules (CBMs), followed by a domain of unknown function, and a catalytic module flanked by a fifth domain. Both Xyn10A CBMs bind calcium ions, and this study explores the effect of these ions on the stability of the full-length enzyme. Xyn10A and truncated forms thereof were produced and their thermostabilities were evaluated under different calcium loads. Studies performed using differential scanning calorimetry showed that the unfolding temperature of the Xyn10A was significantly dependent on the presence of Ca2+, and that the third domain of the enzyme binds at least one Ca2+. Thermal inactivation studies confirmed the role of tightly bound Ca2+ in stabilizing the enzyme, but showed that the presence of a large excess of this ion results in reduced kinetic stability. The truncated forms of Xyn10A were less stable than the full-length enzyme, indicative of module/domain thermostabilizing interactions. Finally, possible roles of the two domains of unknown function are discussed in the light of this study. This is the first report on the thermostabilizing role of calcium on a modular family 10 xylanase that displays multiple calcium binding in three of its five domains/modules.
Abou-Hachem
Maher
M
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, 221 00 Lund, Sweden.
Olsson
Fredrik
F
Nordberg Karlsson
Eva
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D004055:Differential Thermal Analysis; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides; D011485:Protein Binding; D045245:Rhodothermus / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D009841:Oligonucleotides; D014995:Xylosidases; D002118:Calcium