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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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23001 43409 2014 8 PDF Available
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

•Crystal structure of β-galactosidase from Bacillus circulans (BgaC).•Generation of a galactosynthase (His6BgaC-Glu233Gly) by rational design.•The galactosynthase shows a remarkable broad substrate spectrum.•LacNAc type 1 was synthesized for the first time with a galactosynthase.

The crystal structure of β-galactosidase from Bacillus circulans (BgaC) was determined at 1.8 Å 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; Glycosynthase; Glycoconjugates
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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
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 78–85
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
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