4BCE

crystal structure of Ttb-gly N282T mutant

4BCE の概要

• エントリーDOI: 10.2210/pdb4bce/pdb
• 分子名称: BETA-GLUCOSIDASE (2 entities in total)
• 機能のキーワード: hydrolase, glycoside hydrolase family 1, transglycosidase
• 由来する生物種: THERMUS THERMOPHILUS HB8
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 148460.30

構造登録者

Teze, D.,Tran, V.,Tellier, C.,Dion, M.,Leroux, C.,Roncza, J.,Czjzek, M. (登録日: 2012-10-02, 公開日: 2013-03-06, 最終更新日: 2023-12-20)

主引用文献

Teze, D.,Hendrickx, J.,Czjzek, M.,Ropartz, D.,Sanejouand, Y.,Tran, V.,Tellier, C.,Dion, M.
Semi-Rational Approach for Converting a Gh1 Beta-Glycosidase Into a Beta-Transglycosidase.
Protein Eng.Des.Sel., 27:13-, 2014

PubMed Abstract: A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus β-glycosidase (Ttβ-gly, glycoside hydrolase family 1 (GH1)), which display a higher transglycosylation/hydrolysis ratio than the wild-type enzyme. In order to find the cause of these activity modifications, and thereby set up a generic method for easily obtaining transglycosidases from glycosidases, we determined their X-ray structure. No major structural changes could be observed which could help to rationalize the mutagenesis of glycosidases into transglycosidases. However, as these mutations are highly conserved in GH1 β-glycosidases and are located around the -1 site, we pursued the isolation of new transglycosidases by targeting highly conserved amino acids located around the active site. Thus, by single-point mutagenesis on Ttβ-gly, we created four new mutants that exhibit improved synthetic activity, producing disaccharides in yields of 68-90% against only 36% when native Ttβ-gly was used. As all of the chosen positions were well conserved among GH1 enzymes, this approach is most probably a general route to convert GH1 glycosidases into transglycosidases.

PubMed: 24287187
DOI: 10.1093/PROTEIN/GZT057

実験手法

X-RAY DIFFRACTION (2 Å)