title: Rational design of a glycosynthase by the crystal structure of β-galactosidase from Bacillus circulans (BgaC) and its use for the synthesis of N-acetyllactosamine type 1 glycan structures.
abstract: The crystal structure of β-galactosidase from Bacillus circulans (BgaC) was determined at 1.8Å resolution. The overall structure of BgaC consists of three distinct domains, which are the catalytic domain with a TIM-barrel structure and two all-β domains (ABDs). The main-chain fold and steric configurations of the acidic and aromatic residues at the active site were very similar to those of Streptococcus pneumoniae β(1,3)-galactosidase BgaC in complex with galactose. The structure of BgaC was used for the rational design of a glycosynthase. BgaC belongs to the glycoside hydrolase family 35. The essential nucleophilic amino acid residue has been identified as glutamic acid at position 233 by site-directed mutagenesis. Construction of the active site mutant BgaC-Glu233Gly gave rise to a galactosynthase transferring the sugar moiety from α-d-galactopyranosyl fluoride (αGalF) to different β-linked N-acetylglucosamine acceptor substrates in good yield (40-90%) with a remarkably stable product formation. Enzymatic syntheses with BgaC-Glu233Gly afforded the stereo- and regioselective synthesis of β1-3-linked key galactosides like galacto-N-biose or lacto-N-biose.
authors.affiliation: Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, 52074 Aachen, Germany.
authors.forename: Manja
authors.initials: M
authors.lastname: Henze
authors.affiliation: Division of Electron Microscopic Research, Korea Basic Science Institute (KBSI), Daejeon, South Korea; Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka, Japan.
authors.forename: Dong-Ju
authors.initials: DJ
authors.lastname: You
authors.affiliation: Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, 52074 Aachen, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Kamerke
authors.affiliation: Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, Stetternicher Forst Geb. 15.8, 52426 Jülich, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Hoffmann
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka, Japan; International College, Osaka University, 1-30 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Clement
authors.initials: C
authors.lastname: Angkawidjaja
authors.affiliation: Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, 52074 Aachen, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Ernst
authors.affiliation: Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, Stetternicher Forst Geb. 15.8, 52426 Jülich, Germany; IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Pietruszka
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
authors.affiliation: Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, 52074 Aachen, Germany. Electronic address: l.elling@biotec.rwth-aachen.de.
authors.forename: Lothar
authors.initials: L
authors.lastname: Elling
mesh_terms: D000606:Amino Sugars / Q000096:biosynthesis* / Q000737:chemistry; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005690:Galactose / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry; D017510:Protein Folding; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry*
chemical_list: D000606:Amino Sugars; D011134:Polysaccharides; C000458:N-acetyllactosamine; D001616:beta-Galactosidase; D005690:Galactose
keywords: Bacillus circulans β-galactosidase; Crystal structure; Glycoconjugates; Glycosynthase