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

Abstract

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