PEDS Advance Access originally published online on January 11, 2007
Protein Engineering Design and Selection 2007 20(1):15-23; doi:10.1093/protein/gzl049
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New insights into the role of the thumb-like loop in GH-11 xylanases
1 Institut National de la Recherche Agronomique, UMR FARE 614, 8 rue Gabriel Voisin, BP 316, 51688 Reims Cedex 2, France 2 Centre National de la Recherche Scientifique, Université de Nantes, UMR 6204, 2 rue de la Houssinière, 44322 Nantes Cedex 03, France
5 To whom correspondence should be addressed. E-mail: michael.odonohue{at}insa-toulouse.fr
GH-11 xylanases are highly specific and possess a thumb-shaped loop, a unique structure among enzymes with a jelly-roll scaffold. To investigate this structure, in vitro mutagenesis was performed on a GH-11 xylanase (Tx-Xyl) from Thermobacillus xylanilyticus. Targets were the conserved amino acids Pro114-Ser115-Ile116 that are located at the thumb's tip and Thr121 and Tyr111, linker residues that connect the thumb to the main enzyme scaffold. Site-saturation mutagenesis provided an active variant that possesses a new triplet (Pro114-Gly115-Cys116), not found in naturally occurring GH-11 xylanases. The kcat value for xylan hydrolysis catalysed by this mutant was increased by 20%. Re-positioning of the thumb through the deletion of the linker residues produced different effects. As predicted by in silico analyses, deletion of Thr121 had drastic consequences on activity, whereas deletion of Tyr111 only affected (4-fold decrease) kcat. Finally, deletion mutagenesis was used to create a thumbless variant that was almost catalytically inactive. Fluorescence titration with xylotetraose and xylopentaose revealed that this thumb-deleted xylanase retained the ability to bind substrates. This binding was comparable to that of the wild-type enzyme. Additionally, unlike wild-type Tx-Xyl, the thumb-deleted xylanase efficiently bound cellotetraose, although no cellulose hydrolysing activity was detected. Overall, these data show that the thumb is a key determinant for substrate selection and support previous data that suggest that it plays a role in the catalytic process.
Keywords: GH-11 xylanases/in vitro mutagenesis/GH-12 endoglucanases/thumb-like loop
Received June 29, 2006; revised October 5, 2006; accepted October 11, 2006.
3 Current address: Institut National de la Recherche Agronomique, UMR1163 ESIL, CP 925, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France
4 Current address: Institut National de la Recherche Agronomique, UMR 792, INSA, 135 Avenue de Rangueil, 31077 Toulouse Cedex 04, France
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