title: Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity.
abstract: Proteins belonging to glycoside hydrolase family 63 (GH63) are found in bacteria, archaea and eukaryotes. Although the eukaryotic GH63 proteins have been identified as processing α-glucosidase I, the substrate specificities of the bacterial and archaeal GH63 proteins are not clear. Here, we converted a bacterial GH63 enzyme, Escherichia coli YgjK, to a glycosynthase to probe its substrate specificity. Two mutants of YgjK (E727A and D324N) were constructed, and both mutants showed glycosynthase activity. The reactions of E727A with β-D-glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A complexed with the reaction product indicated that the disaccharide bound at the active site was 2-O-α-D-glucopyranosyl-α-D-galactopyranose (Glc12Gal). A comparison of the structures of E727A-Glc12Gal and D324N-melibiose showed that there were two main types of conformation: the open and closed forms. The structure of YgjK adopted the closed form when subsite -1 was occupied by glucose. These results suggest that sugars containing the Glc12Gal structure are the most likely candidates for natural substrates of YgjK.
authors.affiliation: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Miyazaki
authors.forename: Megumi
authors.initials: M
authors.lastname: Ichikawa
authors.forename: Gaku
authors.initials: G
authors.lastname: Yokoi
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Kitano
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishikawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Tonozuka
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008553:Melibiose / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C018301:2-(O-alpha-glucopyranosyl)-galactopyranose; D004187:Disaccharides; D029968:Escherichia coli Proteins; D006027:Glycosides; D011994:Recombinant Proteins; C030364:glucosyl fluoride; D008553:Melibiose; D009699:N-Glycosyl Hydrolases; C497011:YgjK protein, E coli; D005947:Glucose; D005690:Galactose
keywords: (α/α)6 barrel; collagen; glycoside hydrolase family 63; glycosynthase; processing α-glucosidase I