TY - JOUR
T1 - Molecular design of non-leloir furanose-transferring enzymes from an alpha-L-arabinofuranosidase
T2 - A rationale for the engineering of evolved transglycosylases
AU - Bissaro, Bastien
AU - Durand, Julien
AU - Biarnés, Xevi
AU - Planas, Antoni
AU - Monsan, Pierre
AU - O'Donohue, Michael J.
AU - Fauré, Régis
N1 - Publisher Copyright:
© 2015 American Chemical Society.
PY - 2015/8
Y1 - 2015/8
N2 - The vast biodiversity of glycoside hydrolases (GHs) constitutes a reservoir of readily available carbohydrate-acting enzymes that employ simple substrates and hold the potential to perform highly stereopecific and regioselective glycosynthetic reactions. However, most GHs preferentially hydrolyze glycosidic bonds and are thus characterized by a hydrolysis/transglycosylation partition in favor of hydrolysis. Unfortunately, current knowledge is insufficient to rationally modify this partition, specifically mutating key molecular determinants to tip the balance toward transglycosylation. In this study, in the absence of precise knowledge concerning the hydrolysis/transglycosylation partition in a hydrolytic GH51 alpha-L-arabinofuranosidase, we describe how an iterative protein engineering approach has been used to create the first "non-Leloir" transarabinofuranosylases. In the first step, random mutagenesis yielded a point mutation (R69H) at a position that is highly conserved in clan GH-A. Characterization of R69H revealed that this enzyme displays high transglycosylation activity but severely reduced (61-fold) activity on pNP-alpha-L-arabinofuranoside. Upon recombination of R69H with other point mutations selected using semirational or in silico approaches, transfer rates close to 100% and transarabinofuranosylation yields of the main (1 2)-linked oligosaccharide product of 80% (vs 11% for the wild-type) were obtained. Combining data presented here with knowledge drawn from the literature, we suggest that the creation of non-Leloir transglycosylases necessarily involves the destabilization of the highly developed transition states that characterize the predominantly hydrolytic exo-acting GHs; this is an efficient way to prevent ubiquitous water molecules from performing the deglycosylation step.
AB - The vast biodiversity of glycoside hydrolases (GHs) constitutes a reservoir of readily available carbohydrate-acting enzymes that employ simple substrates and hold the potential to perform highly stereopecific and regioselective glycosynthetic reactions. However, most GHs preferentially hydrolyze glycosidic bonds and are thus characterized by a hydrolysis/transglycosylation partition in favor of hydrolysis. Unfortunately, current knowledge is insufficient to rationally modify this partition, specifically mutating key molecular determinants to tip the balance toward transglycosylation. In this study, in the absence of precise knowledge concerning the hydrolysis/transglycosylation partition in a hydrolytic GH51 alpha-L-arabinofuranosidase, we describe how an iterative protein engineering approach has been used to create the first "non-Leloir" transarabinofuranosylases. In the first step, random mutagenesis yielded a point mutation (R69H) at a position that is highly conserved in clan GH-A. Characterization of R69H revealed that this enzyme displays high transglycosylation activity but severely reduced (61-fold) activity on pNP-alpha-L-arabinofuranoside. Upon recombination of R69H with other point mutations selected using semirational or in silico approaches, transfer rates close to 100% and transarabinofuranosylation yields of the main (1 2)-linked oligosaccharide product of 80% (vs 11% for the wild-type) were obtained. Combining data presented here with knowledge drawn from the literature, we suggest that the creation of non-Leloir transglycosylases necessarily involves the destabilization of the highly developed transition states that characterize the predominantly hydrolytic exo-acting GHs; this is an efficient way to prevent ubiquitous water molecules from performing the deglycosylation step.
KW - biocatalysis
KW - enzyme mechanism
KW - glycoside hydrolase
KW - molecular evolution
KW - pentose-based oligosaccharides
KW - transglycosylation
UR - https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=pure_univeritat_ramon_llull&SrcAuth=WosAPI&KeyUT=WOS:000359395100014&DestLinkType=FullRecord&DestApp=WOS
UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-84938717744&origin=inward
U2 - 10.1021/acscatal.5b00949
DO - 10.1021/acscatal.5b00949
M3 - Article
AN - SCOPUS:84938717744
SN - 2155-5435
VL - 5
SP - 4598
EP - 4611
JO - ACS Catalysis
JF - ACS Catalysis
IS - 8
ER -