|Gene:||neuA||Accession Number: G-11115 (MetaCyc)|
Species: Escherichia coli K1
The subunit structure of this enzyme has not been firmly established. The enzyme purified from cell extracts showed a band at 50 kDa on SDS-PAGE [Vann87]. Recombinant enzyme was determined to be active as a monomer, but may exist as a dimer, or higher aggregates under some in vitro conditions [Zapata89].
This enzyme found in eukaryotes and prokaryotes has an important role in activation of sialic acids to their CMP-derivatives for use as a donor in sialyltransferase reactions. The bacterial enzyme is of interest for use in rational drug design strategies (reviewed in [Mizanur08a]).
The native enzyme was purified from the cytoplasmic fraction of Escherichia coli K1 [Vann87]. Recombinant enzyme has been overexpressed, highly purified and used in the enzymatic synthesis of CMP-N-acetyl-β-neuraminate on a multigram scale [Shames91]. Site-directed mutagenesis studies have identified amino acid residues in the active site that are important for catalysis [Stoughton99].
Molecular Weight of Polypeptide: 48.736 kD (from nucleotide sequence), 50.0 kD (experimental) [Vann87 ]
|Cellular Component:||GO:0005829 - cytosol [Vann87]|
Enzymatic reaction of: cytidine 5'-monophosphate N-acetylneuraminate synthetase
Synonyms: cytidine 5'-monophosphate N-acetylneuraminic acid synthetase, N-acylneuraminate cytidylyltransferase, CMP-N-acetylneuraminic acid synthetase, CMP-NeuNAc synthetase, CMP-sialic acid synthetase, cytidine monophosphate sialic acid synthetase
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.
The reaction is favored in the direction shown.
The enzyme catalyzes a nucleophilic attack of the anomeric oxygen of β-N-acetylneuraminate on the α-phosphate of CTP using a divalent cation as cofactor, producing diphosphate and CMP-N-acetyl-β-neuraminate (reviewed in [Mizanur08a]).
The enzyme required a divalent cation, with Mg2+ giving optimal activity. Ca2+, Cd2+ and Zn2+ could not substitute. Several substrate analogs tested, but none were substrates. Periodate-oxidized CTP and 5-mercuri-CTP inhibited the enzyme [Vann87].
pH(opt): 9-10 [Vann87]
Cox02: Cox AD, Hood DW, Martin A, Makepeace KM, Deadman ME, Li J, Brisson JR, Moxon ER, Richards JC (2002). "Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus influenzae." Eur J Biochem 269(16);4009-19. PMID: 12180977
Hood99: Hood DW, Makepeace K, Deadman ME, Rest RF, Thibault P, Martin A, Richards JC, Moxon ER (1999). "Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization." Mol Microbiol 33(4);679-92. PMID: 10447878
Severi05: Severi E, Randle G, Kivlin P, Whitfield K, Young R, Moxon R, Kelly D, Hood D, Thomas GH (2005). "Sialic acid transport in Haemophilus influenzae is essential for lipopolysaccharide sialylation and serum resistance and is dependent on a novel tripartite ATP-independent periplasmic transporter." Mol Microbiol 58(4);1173-85. PMID: 16262798
Severi08: Severi E, Muller A, Potts JR, Leech A, Williamson D, Wilson KS, Thomas GH (2008). "Sialic acid mutarotation is catalysed by the Escherichia coli beta -propeller protein YJHT." J Biol Chem 283(8):4841-9. PMID: 18063573
Shames91: Shames SL, Simon ES, Christopher CW, Schmid W, Whitesides GM, Yang LL (1991). "CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives." Glycobiology 1(2);187-91. PMID: 1823161
Stoughton99: Stoughton DM, Zapata G, Picone R, Vann WF (1999). "Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase." Biochem J 343 Pt 2;397-402. PMID: 10510306
Vann87: Vann WF, Silver RP, Abeijon C, Chang K, Aaronson W, Sutton A, Finn CW, Lindner W, Kotsatos M (1987). "Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase." J Biol Chem 262(36);17556-62. PMID: 2826425
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