Mutagenesis of Catalytic Nucleophile of β‐Galactosidase Retains Residual Hydrolytic Activity and Affords a Transgalactosidase

Abstract

Glycosidases that cleave oligosaccharides can also synthesize the glycosidic bond. Site-directed mutagenesis of the catalytic nucleophile commonly abolishes their hydrolytic activity, affording glycosynthases that use glycosyl fluorides as substrates.Here, the synthetic ability of β-galactosidase from Bacillus circulans isoform A (BgaD-A; EC 3.2.1.23, GH2) was investigated by site-directed mutagenesis. The cold-shock expression ensured selective induction and correct folding. Three mutants were constructed at the active-site catalytic nucleophile E532 as putative glycosynthases. However, none of the mutants could process α-galactosyl fluoride as a galactosyl donor. With only negligible hydrolytic activity, two mutants selectively synthesized azido-functionalized N-acetyllactosamine using the pnitrophenyl β-d-galactoside as a galactosyl donor. Thus, they behaved as transglycosidases. This study demonstrates that substitution at the catalytic nucleophile for the assembly of glycosynthases is not unrestrictedly versatile and that the effect of mutagenesis on synthetic abilities depends on the relative orientations of amino acids in the enzyme active site.

Advanced high-affinity glycoconjugate ligands of galectins

“…For structural analysis see the Supporting Information, Table S2, Figure S3a-S3d for compound 3; Table S3, Figure S4a-S4d for compound 4. (6,7,8) Compound 6 (LacNAc-N 3 , β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-Dglucopyranosyl azide) was synthesized as described previously [31]. Briefly, the glycosyl donor pNP-β-Gal (5; 30 mM, 36 mg) and the acceptor GlcNAc-N 3 (1; prepared as described previously [30]; 150 mM, 148 mg) were suspended in 50 mM sodium phosphate buffer pH 6.0, and β-galactosidase BgaD-A (252 µg, 2.4 U, 260 µL) was added.…”

“…The synthesis of ligand 6 (LacNAc-N 3 ) was essentially performed as described previously [31]. For the galactosylation reaction, pNP-β-Gal (2) was used as a glycosyl donor and GlcNAc-N 3 (1) as an acceptor.…”

“…For the galactosylation reaction, pNP-β-Gal (2) was used as a glycosyl donor and GlcNAc-N 3 (1) as an acceptor. The enzymatic synthesis was catalyzed by β-galactosidase from B. circulans [31] and afforded 6 in 15% yield. Alternatively, the same reaction with the synthetically potent mutant E532G BgaD-A afforded a higher yield of 6 (44%) [31].…”

Mutation Hotspot for Changing the Substrate Specificity of β-N-Acetylhexosaminidase: A Library of GlcNAcases

“…Notably, in many prepared variants, the significant increase in selectivity (GalNAcase/GlcNAcase activity ratio decreased from 3- to 8-fold compared to WT) has not caused a considerable decline in specific activity as is otherwise usual in active site mutant variants, particularly those at the catalytic nucleophile [ 51 ]. Here, in many cases, we rather observed an increase in the enzyme specific activity compared with the WT.…”

Enzymatic Glycosylation Strategies in the Production of Bioactive Compounds

“…Given the versatility of β-galactosidase from Bacillus circulans mutant (BgaC), P. Bojarová et al [73] reported the synthetic application of these mutant enzymes for the transformation of α-galactosyl fluoride (αGF) and β-galactosyl azide (βGN3) α-galactosyl to test the glycosynthase activity. Those three different mutants were obtained using selective mutagenesis, via active site modification to glycine, alanine, and threonine, and they were then applied in the transformation.…”