The wciN gene encodes an α-1,3-galactosyltransferase involved in the biosynthesis of the capsule repeating unit of Streptococcus pneumoniae serotype 6B.

Almost all Streptococcus pneumoniae (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 wciN gene was predicted to encode a galactosyltransferase involved in the RU assembly of pneumococcus type 6B CPS. Herein, we provide the unambiguous in vitro biochemical evidence that wciN encodes an alpha-1,3-galactosyltransferase catalyzing the transfer of galactosyl from UDP-Gal onto the Glc alpha-pyrophosphate-lipid (Glc alpha-PP-lipid) acceptor to form Gal alpha(1-3)Glc alpha-PP-lipid. A chemically synthesized acceptor (Glc alpha-PP-O(CH2)(10)CH3) was used to characterize the WciN activity. The disaccharide product, i.e., Gal alpha(1-3)Glc alpha-PP-O(CH2)(10)CH3, 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 in vitro and will help to better understand the pneumococcal CPS biosynthetic pathway.