Directed evolution of a β-glycosidase from Agrobacterium sp. to enhance its glycosynthase activity toward C3-modified donor sugars.
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 'bio-orthogonal tags'. 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. β-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 β-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-β-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.
Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Jae-Hoon
JH
Shim
Hong-Ming
HM
Chen
Jamie R
JR
Rich
Ethan D
ED
Goddard-Borger
Stephen G
SG
Withers
D019325:3-O-Methylglucose / Q000378:metabolism*; D060054:Agrobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D019020:Directed Molecular Evolution / Q000379:methods*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D019325:3-O-Methylglucose; D001617:beta-Glucosidase