title: The wciN gene encodes an α-1,3-galactosyltransferase involved in the biosynthesis of the capsule repeating unit of Streptococcus pneumoniae serotype 6B.
abstract: 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 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(CH(2))(10)CH(3)) was used to characterize the WciN activity. The disaccharide product, i.e., Galα(1-3)Glcα-PP-O(CH(2))(10)CH(3), 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.
authors: State Key Laboratory of Medicinal Chemical Biology,  Nankai University , Tianjin 300071, China.
authors: Weiqing
authors: W
authors: Han
authors: Li
authors: L
authors: Cai
authors: Baolin
authors: B
authors: Wu
authors: Lei
authors: L
authors: Li
authors: Zhongying
authors: Z
authors: Xiao
authors: Jiansong
authors: J
authors: Cheng
authors: Peng G
authors: PG
authors: Wang
mesh_terms: D000595:Amino Acid Sequence; D016667:Bacterial Capsules / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
chemical_list: D008345:Manganese; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase; D008274:Magnesium; D015032:Zinc