Expression of alpha2,8/2,9-polysialyltransferase from Escherichia coli K92. Characterization of the enzyme and its reaction products.
The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAcalpha2- and -9-NeuAcalpha2- 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 alpha2,9- and alpha2,8-linked polysialyl structure. The K92 neuS gene was used to transform the K1 strain of E. coli, the capsule of which contains only -8-NeuAcalpha2- linkages. Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAcalpha2- and -9-NeuAcalpha2- linkages. Our results suggest that the neuS gene product of E. coli K92 catalyzes the synthesis of polysialic acid with alpha2,9- and alpha2,8-linkages in vitro and in vivo.
Shen
G J
GJ
Department of Chemistry, Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Datta
A K
AK
Izumi
M
M
Koeller
K M
KM
Wong
C H
CH
D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014170:Transformation, Genetic
D029968:Escherichia coli Proteins; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; C403313:NeuS protein, E coli; D012799:Sialyltransferases