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J Biol Chem, Vol. 274, Issue 49, 35139-35146, December 3, 1999

Expression of 2,8/2,9-Polysialyltransferase from Escherichia coli K92
CHARACTERIZATION OF THE ENZYME AND ITS REACTION PRODUCTS

From the Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037

The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAc2- and -9-NeuAc2- 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 2,9- and 2,8-linked polysialyl structure. The K92 neuS gene was used to transform the K1 strain of E. coli, the capsule of which contains only -8-NeuAc2- linkages. Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAc2- and -9-NeuAc2- linkages. Our results suggest that the neuS gene product of E. coli K92 catalyzes the synthesis of polysialic acid with 2,9- and 2,8-linkages in vitro and in vivo.

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