Semi-rational approach for converting a GH1 β-glycosidase into a β-transglycosidase.
2014
Teze, David | Hendrickx, Johann | Czjzek, Mirjam | Ropartz, David | Sanejouand, Yves-Henri | Tran, Vinh | Tellier, Charles | Dion, Michel | Laboratoire de Biologie Intégrative des Modèles Marins (LBI2M) ; Station biologique de Roscoff [Roscoff] (SBR) ; Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS) | Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA) ; Institut National de la Recherche Agronomique (INRA) | Végétaux marins et biomolécules ; Station biologique de Roscoff [Roscoff] (SBR) ; Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-GOEMAR-Centre National de la Recherche Scientifique (CNRS) | Unité de Biotechnologie, Biocatalyse et Biorégulation (U3B) ; Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST) ; Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS) | Glyconet Network
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Показать больше [+] Меньше [-]Английский. 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.
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