Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.
N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid. We have purified CMP-NeuAc synthetase from an Escherichia coli O18:K1 cytoplasmic fraction to apparent homogeneity by ion exchange chromatography and affinity chromatography on CDP-ethanolamine linked to agarose. The enzyme has a specific activity of 2.1 mumol/mg/min and migrates as a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis. The enzyme has a requirement for Mg2+ or Mn2+ and exhibits optimal activity between pH 9.0 and 10. The apparent Michaelis constants for the CTP and NeuAc are 0.31 and 4 mM, respectively. The CTP analogues 5-mercuri-CTP and CTP-2',3'-dialdehyde are inhibitors. The purified CMP-N-acetylneuraminic acid synthetase has a molecular weight of approximately 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding CMP-N-acetylneuraminic acid synthetase is located on a 3.3-kilobase HindIII fragment. The purified enzyme appears to be identical to the 50,000 Mr polypeptide encoded by this gene based on insertion mutations that result in the loss of detectable enzymatic activity. The amino-terminal sequence of the purified protein was used to locate the start codon for the CMP-NeuAc synthetase gene. Both the enzyme and the 50,000 Mr polypeptide have the same NH2-terminal amino acid sequence. Antibodies prepared to a peptide derived from the NH2-terminal amino acid sequence bind to purified CMP-NeuAc synthetase.
Vann
W F
WF
Laboratory of Bacterial Polysaccharides, Food and Drug Administration, Bethesda, Maryland 20892.
Silver
R P
RP
Abeijon
C
C
Chang
K
K
Aaronson
W
W
Sutton
A
A
Finn
C W
CW
Lindner
W
W
Kotsatos
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D000216:N-Acylneuraminate Cytidylyltransferase / Q000235:genetics / Q000302:isolation & purification*; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D010957:Plasmids
D000596:Amino Acids; D008345:Manganese; D008715:Methionine; D009713:Nucleotidyltransferases; D000216:N-Acylneuraminate Cytidylyltransferase; D008274:Magnesium