Expression of α2,8/2,9-Polysialyltransferase fromEscherichia coli K92

• Published in: The Journal of biological chemistry 1999-12, Vol.274 (49), p.35139
• Main Authors: Gwo-Jenn Shen, Arun K. Datta, Masayuki Izumi, Kathryn M. Koeller, Chi-Huey Wong
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.49.35139

The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAcα2- and -9-NeuAcα2- linkages. The enzyme catalyzing this polymerizing reaction has been cloned from the genomic DNA of E. coli K92. The 1.2-kilobase polymerase chain reaction fragment was subcloned in pRSET vector and the protein was expressed in the BL21(DE3) strain of E. coli with a hexameric histidine at its N-terminal end. The enzyme was isolated in the supernatant after lysis of the cells and fractionated by ultracentrifugation. Western blotting using anti-histidine antibody showed the presence of a band that migrated at about 47.5 kDa on both reducing and nonreducing SDS-polyacrylamide gel electrophoresis, indicating a monomeric enzyme. Among the carbohydrate acceptors tested, N -acetylneuraminic acid and the gangliosides G D3 and G Q1b were preferred substrates. The cell-free enzyme reaction products obtained were characterized by NMR and mass spectrometry, which indicated the presence of both α2,9- and α2,8-linked polysialyl structure. The K92 neuS gene was used to transform the K1 strain of E. coli , the capsule of which contains only -8-NeuAcα2- linkages. Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAcα2- and -9-NeuAcα2- linkages. Our results suggest that the neuS gene product of E. coli K92 catalyzes the synthesis of polysialic acid with α2,9- and α2,8-linkages in vitro and in vivo.