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350 a.a.
_MG
Key reference
DOI no: 10.1016/j.jmb.2006.05.002 J Mol Biol 360:157-167 (2006) PubMed id: 16762367
Probing the structural basis for the difference in thermostability displayed by family 10 xylanases. H.Xie, J.Flint, M.Vardakou, J.H.Lakey, R.J.Lewis, H.J.Gilbert, C.Dumon. ABSTRACT
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 degrees C higher than the wild-type enzyme and, at 55 degrees C, the first-order rate constant for thermal inactivation of A334V/G348D is 4.1 x 10(-4) min(-1), compared to a value of 1.6 x 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 approximately 2 degrees 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 O(delta1) of Asp348 makes an additional hydrogen bond with the amide of Gly344 and O(delta2) 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.
Selected figure(s)
Figure 1.
Figure 1. Thermostability of CmXyn10B and its derivatives at different temperatures. CmXyn10B was incubated for 15 min at various temperatures and assayed for residual PNPCase activity at 37 °C.
Figure 4.
Figure 4. The crystal structure of the CmXyn10B derivative A334V/G348D. (a) The location of A334V and G348V in CmXyn10B. (b) and (c) The interactions made by Val334 and Asp348, respectively in the A334V/G348D mutant. In (b), the wild-type protein (in green) is overlaid with the A334V/G348D mutant (blue), while the bound reaction product (AX[2]) is displayed in yellow. The Figure was prepared using PyMOL (http://www.pymol.sourceforge.net/).
The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 360, 157-167) copyright 2006. Figures were selected by an automated process.
Literature references that cite this PDB file's key reference
PubMed id Reference
18725971 A.Bharadwaj, S.Leelavathi, S.Mazumdar-Leighton, A.Ghosh, S.Ramakumar, and V.S.Reddy (2008).
The critical role of partially exposed N-terminal valine residue in stabilizing GH10 xylanase from Bacillus sp.NG-27 under poly-extreme conditions.PLoS ONE, 3, e3063.
18515360 C.Dumon, A.Varvak, M.A.Wall, J.E.Flint, R.J.Lewis, J.H.Lakey, C.Morland, P.Luginbühl, S.Healey, T.Todaro, G.DeSantis, M.Sun, L.Parra-Gessert, X.Tan, D.P.Weiner, and H.J.Gilbert (2008).
Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure.J Biol Chem, 283, 22557-22564.
PDB codes: 2vuj 2vul The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.
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