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

The crystal structure of p-galactosidase from Bacillus circulans (BgaC) was determined at 1.8 A resolution. The overall structure of BgaC consists of three distinct domains, which are the catalytic domain with a TIM-barrel structure and two all-p 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 p(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-G1u233Gly gave rise to a galactosynthase transferring the sugar moiety from alpha-D-galactopyranosyl fluoride (alpha GalF) to different beta-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 beta 1-3-linked key galactosides like galactoN-biose or lacto-N-biose. (C) 2014 Published by Elsevier B.V.