Anatomy of glycosynthesis: structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants.
The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).
Ducros
Valérie M-A
VM
Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York YO10 5YW, United Kingdom.
Tarling
Chris A
CA
Zechel
David L
DL
Brzozowski
A Marek
AM
Frandsen
Torben P
TP
von Ossowski
Ingemar
I
Schülein
Martin
M
Withers
Stephen G
SG
Davies
Gideon J
GJ
D002475:Cellobiose / Q000378:metabolism; D003460:Crystallization; D003627:Data Interpretation, Statistical; D005658:Fungi / Q000201:enzymology / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006027:Glycosides / Q000096:biosynthesis*; D007202:Indicators and Reagents; D007700:Kinetics; D007785:Lactose / Q000031:analogs & derivatives*; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism
D006027:Glycosides; D007202:Indicators and Reagents; D009844:Oligosaccharides; C084793:lactosyl fluoride; D002475:Cellobiose; D006026:Glycoside Hydrolases; D007785:Lactose