@article { ISI:000308232000004,
	title = {Directed evolution of a beta-glycosidase from Agrobacterium sp. to enhance its glycosynthase activity toward C3-modified donor sugars},
	journal = {PROTEIN ENGINEERING DESIGN \& SELECTION},
	volume = {25},
	number = {9},
	year = {2012},
	month = {SEP},
	pages = {465-472},
	abstract = {Glycans bearing modified hydroxyl groups are common in biology but because these modifications are added after assembly, enzymes are not available for the transfer and coupling of hydroxyl-modified monosaccharide units. Access to such enzymes could be valuable, particularly if they can also introduce obio-orthogonal tags{\textquoteright}. Glycosynthases, mutant glycosidases that synthesize glycosides using glycosyl fluoride donors, are a promising starting point for creation of such enzymes through directed evolution. Inspection of the active site of a homology model of the GH1 Agrobacterium sp. -glycosidase, which has both glucosidase and galactosidase activity, identified Q24, H125, W126, W404, E411 and W412 as amino acids that constrain binding around the 3-OH group, suggesting these residues as targets for mutation to generate an enzyme capable of handling 3-O-methylated sugars. Site-directed saturation mutagenesis at these positions within the wild-type -glycosidase gene and screening via an on-plate assay yielded two mutants (Q24S/W404L and Q24N/W404N) with an improved ability to hydrolyze 4-nitrophenyl 3-O-methyl{\textendash}d-galactopyranoside (3-MeOGal-pNP). Translation of these mutations into the evolved glycosynthase derived from the same glucosidase (2F6) yielded glycosynthases (AbgSL-T and AbgNN-T, where T denotes transferase) capable of forming 3-O-methylated glucosides on multi-milligram scales at rates approximately 5 and 40 times greater, respectively, than the parent glycosynthase.},
	issn = {1741-0126},
	doi = {10.1093/protein/gzs045},
	author = {Shim, Jae-Hoon and Chen, Hong-Ming and Rich, Jamie R. and Goddard-Borger, Ethan D. and Withers, Stephen G.}
}
