title: Transglycosylation of engineered cyclodextrin glucanotransferases as O-glycoligases.
abstract: An O-glycoligase is a hydrolytically impaired mutant of a retaining α-glycosidase in which the catalytic acid/base has been removed, but which can still perform transglycosylation when incubated with activated glycosyl fluoride donor sugars. In this paper, we describe another example, wherein a cyclodextrin glucanotransferase mutant (CGT-E284A) with an alanine residue at its general acid/base catalyst position (Glu284), was constructed. This mutant was hydrolytically inactive, but exhibited significant transglycosylation activity using α-maltosyl fluoride (αG2F) as donor, and either 4-nitrophenyl glucosides or maltosides as acceptors. To improve transglycosylation activity, a site-saturation mutagenesis library at Glu284 was created. Through a thin-layer chromatography-based screening process, two mutants were identified; (1) a mutant with a glycine residue at Glu284 (CGT-E284G) exhibiting improved transglycosylation activity compared with the original alanine mutant and (2) a mutant with a serine residue with residual hydrolytic activity. Kinetic analysis revealed that 4-nitrophenyl maltosides were better acceptors than 4-nitrophenyl glucosides. Transglycosylation activities of CGT-E284A and CGT-E284G were inhibited at high concentrations (>0.8 mM) of the acceptor sugars. In contrast, typical saturation kinetic behavior was observed upon varying the donor (αG2F) concentration at a fixed acceptor concentration (0.8 mM). The catalytic efficiencies (apparent k(cat)/K(M)) of CGT-E284G were generally three- to sixfold higher than those of CGT-E284A. Due to the rate at high concentrations of the acceptors, higher transglycosylation yields were achieved at a low concentration of the acceptors (69-84% at 1 mM) compared to those at a higher concentration (22-36% at 10mM).
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong, 339-700, Republic of Korea.
authors.forename: Chao
authors.initials: C
authors.lastname: Li
authors.forename: Hee-Jeong
authors.initials: HJ
authors.lastname: Ahn
authors.forename: Jin-Hyo
authors.initials: JH
authors.lastname: Kim
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
mesh_terms: D000409:Alanine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005960:Glucosides / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering; D012694:Serine / Q000378:metabolism
chemical_list: D005960:Glucosides; C036988:4-nitrophenylmaltoside; C019502:4-nitrophenyl alpha-glucoside; D018698:Glutamic Acid; D012694:Serine; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D008025:Ligases; D000409:Alanine
keywords: Cyclodextrin glucanotransferase; O-Glycoligase; O-α-Glycosidic linkage; Saturation mutagenesis; Transglycosylation