Probing the structural basis for the difference in thermostability displayed by family 10 xylanases

Xie, Hefang, Flint, James, Vardakou, Maria , Lakey, Jeremy H., Lewis, Richard J., Gilbert, Harry J. and Dumon, Claire (2006) Probing the structural basis for the difference in thermostability displayed by family 10 xylanases. Journal of Molecular Biology, 360 (1). pp. 157-167. ISSN 0022-2836

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Thermostability is an important property of industrially significant hydrolytic enzymes: understanding the structural basis for this attribute will underpin the future biotechnological exploitation of these biocatalysts. The Cellvibrio family 10 (GH10) xylanases display considerable sequence identity but exhibit significant differences in thermostability; thus, these enzymes represent excellent models to examine the structural basis for the variation in stability displayed by these glycoside hydrolases. Here, we have subjected the intracellular Cellvibrio mixtus xylanase CmXyn10B to forced protein evolution. Error-prone PCR and selection identified a double mutant, A334V/G348D, which confers an increase in thermostability. The mutant has a Tm 8 °C higher than the wild-type enzyme and, at 55 °C, the first-order rate constant for thermal inactivation of A334V/G348D is 4.1 � 10-4 min-1, compared to a value of 1.6 � 10-1 min-1 for the wild-type enzyme. The introduction of the N to C-terminal disulphide bridge into A334V/G348D, which increases the thermostability of wild-type CmXyn10B, conferred a further �2 °C increase in the Tm of the double mutant. The crystal structure of A334V/G348D showed that the introduction of Val334 fills a cavity within the hydrophobic core of the xylanase, increasing the number of van der Waals interactions with the surrounding aromatic residues, while Oδ1 of Asp348 makes an additional hydrogen bond with the amide of Gly344 and Oδ2 interacts with the arabinofuranose side-chain of the xylose moiety at the -2 subsite. To investigate the importance of xylan decorations in productive substrate binding, the activity of wild-type CmXyn10B, the mutant A334V/G348D, and several other GH10 xylanases against xylotriose and xylotriose containing an arabinofuranose side-chain (AX3) was assessed. The enzymes were more active against AX3 than xylotriose, providing evidence that the arabinose side-chain makes a generic contribution to substrate recognition by GH10 xylanases. © 2006 Elsevier Ltd. All rights reserved.

Keywords:arabinose derivative, xylan, xylan endo 1,3 beta xylosidase, xylose, amino acid sequence, amino terminal sequence, article, carboxy terminal sequence, Cellvibrio, controlled study, enzyme activity, enzyme inactivation, enzyme stability, enzyme structure, enzyme substrate complex, hydrophobicity, nonhuman, polymerase chain reaction, priority journal, protein family, structure analysis, temperature dependence, thermostability, wild type, Biochemistry, Crystallography, X-Ray, Endo-1,4-beta Xylanases, Glycoside Hydrolases, Guanidine, Hydrolysis, Models, Molecular, Mutagenesis, Site-Directed, Plasmids, Protein Conformation, Protein Denaturation, Temperature, Thermodynamics, Cellvibrio, Cellvibrio mixtus
Subjects:C Biological Sciences > C720 Biological Chemistry
Divisions:College of Science > School of Life Sciences
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ID Code:12469
Deposited On:16 Dec 2013 14:59

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