title: Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire.
abstract: The Agrobacterium sp. beta-glucosidase (Abg) is a retaining beta-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using alpha-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using alpha-D-galactopyranosyl fluoride and 4-nitrophenyl beta-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.
authors.affiliation: Protein Engineering Network of Centres of Excellence of Canada, British Columbia, Canada.
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
authors.forename: Seung Seo
authors.initials: SS
authors.lastname: Lee
authors.forename: R Antony J
authors.initials: RA
authors.lastname: Warren
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D001665:Binding Sites; D001669:Biochemical Phenomena; D001671:Biochemistry; D019020:Directed Molecular Evolution / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000737:chemistry; D015723:Gene Library; D005822:Genetic Vectors; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport; D012231:Rhizobium / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics
chemical_list: C074869:7-deoxy-13-dihydroadriamycinone; D018698:Glutamic Acid; D004317:Doxorubicin; D014364:Tryptophan; D001617:beta-Glucosidase