A β-(1,2)-glycosynthase and an attempted selection method for the directed evolution of glycosynthases.
Understanding how enzymes mediate catalysis is a key to their reprogramming for biotechnological applications. The family 3 retaining glycosidase postulated to be involved in erythromycin self-resistance was cloned, recombinantly expressed in Escherichia coli, purified, and characterized. Bioinformatics analysis allowed the identification of the acid/base and nucleophile residues, and mutation of these residues resulted in hydrolytically inactive proteins. One mutant was able to synthesize a glycosidic linkage using α-glucosyl fluoride as a donor and macrolide antibiotics as acceptors. This shows an unprecedented application of glycosynthase technology in accomplishing a challenging β-(1,2)-glycosylation of an amino sugar. This work also provides the first biochemical characterization of the EryBI protein and supports its role in the self-resistance mechanism involved in erythromycin biosynthesis. An in vivo selection approach was used in an attempt to spur evolution of the glycosynthase, and the results from the attempted selection method provide insight into the requirements for in vivo directed evolution of glycosynthases.
College of Pharmacy, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada. david.jakeman@dal.ca
David L
DL
Jakeman
Ali
A
Sadeghi-Khomami
D000606:Amino Sugars / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D004917:Erythromycin / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D009154:Mutation; D016930:Saccharopolyspora / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D000606:Amino Sugars; D001426:Bacterial Proteins; D004917:Erythromycin; D006026:Glycoside Hydrolases