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.
Keywords: Bacillus circulans β-galactosidase; Crystal structure; Glycoconjugates; Glycosynthase.
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