The <i>wciN</i> Gene Encodes an α-1,3-Galactosyltransferase Involved in the Biosynthesis of the Capsule Repeating Unit of <i>Streptococcus pneumoniae</i> Serotype 6B

Almost all <i>Streptococcus pneumoniae</i> (pneumococcus) capsule serotypes employ the Wzy-dependent pathway for their capsular polysaccharide (CPS) biosynthesis. The assembly of the CPS repeating unit (RU) is the first committed step in this pathway. The <i>wciN</i> gene was predicted to encode a galactosyltransferase involved in the RU assembly of pneumococcus type 6B CPS. Herein, we provide the unambiguous <i>in vitro</i> biochemical evidence that <i>wciN</i> encodes an α-1,3-galactosyltransferase catalyzing the transfer of galactosyl from UDP-Gal onto the Glcα-pyrophosphate-lipid (Glcα-PP-lipid) acceptor to form Galα(1–3)­Glcα-PP-lipid. A chemically synthesized acceptor (Glcα-PP-O­(CH₂)₁₀CH₃) was used to characterize the WciN activity. The disaccharide product, i.e., Galα(1–3)­Glcα-PP-O­(CH₂)₁₀CH₃, was characterized by mass and NMR spectroscopy. Substrate specificity study indicated that the acceptor structural region composed of pyrophosphate and lipid moieties may play an important role in the enzyme-acceptor recognition. Furthermore, divalent metal cations were found indispensable to the WciN activity, suggesting that this glycosyltransferase (GT) belongs to the GT-A superfamily. By analyzing the activities of six WciN mutants, a DXD motif involved in the coordination of a divalent metal cation was identified. This work provides a chemical biology approach to characterize the activities of pneumococcal CPS GTs <i>in vitro</i> and will help to better understand the pneumococcal CPS biosynthetic pathway

Regioselective Chemoenzymatic Synthesis of Ganglioside Disialyl Tetrasaccharide Epitopes

A novel chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes found as the terminal oligosaccharides of GD1α, GT1aα, and GQ1bα is described. It relies on chemical manipulation of enzymatically generated trisaccharides as conformationally constrained acceptors for regioselective enzymatic α2–6-sialylation. This strategy provides a new route for easy access to disialyl tetrasaccharide epitopes and their derivatives

Successfully Engineering a Bacterial Sialyltransferase for Regioselective α2,6-sialylation

A β-galactoside α2,6-sialyltransferase from <i>Photobacterium damselae</i> (Pd2,6ST) that is capable of sialylating both terminal and internal galactose and <i>N</i>-acetylgalactosamine was herein redesigned for regioselectively producing terminal α2,6-sialosides. Guided by a recently developed bump-hole strategy, a series of mutations at Ala200 and Ser232 sites were created for reshaping the acceptor binding pocket. Finally, a Pd2,6ST double mutant A200Y/S232Y with an altered L-shaped acceptor binding pocket was identified to be a superior α2,6-sialyltransferase which can efficiently catalyze the regioselective α2,6-sialylation of galactose or <i>N</i>-acetylgalactosamine at the nonreducing end of a series of glycans. Meanwhile, A200Y/S232Y remains flexible donor substrate specificity and is able to transfer Neu5Ac, Neu5Gc, and KDN

Site-Directed Glycosylation of Peptide/Protein with Homogeneous O‑Linked Eukaryotic N‑Glycans

Here we report a facile and efficient method for site-directed glycosylation of peptide/protein. The method contains two sequential steps: generation of a GlcNAc-O-peptide/protein, and subsequent ligation of a eukaryotic N-glycan to the GlcNAc moiety. A pharmaceutical peptide, glucagon-like peptide-1 (GLP-1), and a model protein, bovine α-Crystallin, were successfully glycosylated using such an approach. It was shown that the GLP-1 with O-linked N-glycan maintained an unchanged secondary structure after glycosylation, suggesting the potential application of this approach for peptide/protein drug production. In summary, the coupled approach provides a general strategy to produce homogeneous glycopeptide/glycoprotein bearing eukaryotic N-glycans