New insights into the role of the thumb-like loop in GH-11 xylanases - INRAE - Institut national de recherche pour l’agriculture, l’alimentation et l’environnement Access content directly
Journal Articles Protein Engineering, Design and Selection Year : 2007

New insights into the role of the thumb-like loop in GH-11 xylanases

Abstract

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 Pro(114)-Ser(115)-Ile(116) that are located at the thumb's tip and Thr(121) and Tyr(111), linker residues that connect the thumb to the main enzyme scaffold. Site-saturation mutagenesis provided an active variant that possesses a new triplet (Pro(114)-Gly(115)-Cys(116)), not found in naturally occurring GH-11 xylanases. The k(cat) 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 Thr(121) had drastic consequences on activity, whereas deletion of Tyr(111) only affected (4-fold decrease) k(cat). 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.

Dates and versions

hal-02659467 , version 1 (30-05-2020)

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Gabriel Paës, Vinh Tran, Masayuki Takahashi, Imen Boukari, Michael O Donohue. New insights into the role of the thumb-like loop in GH-11 xylanases. Protein Engineering, Design and Selection, 2007, 20 (1), pp.15-23. ⟨10.1093/protein/gzl049⟩. ⟨hal-02659467⟩
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