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MetaCyc Enzyme: cytidine 5'-monophosphate N-acetylneuraminate synthetase

Gene: neuA Accession Number: G-11115 (MetaCyc)

Species: Escherichia coli K1

Summary:
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]).

In the pathogen Escherichia coli K1 the capsular polysaccharide is a homopolymer of α-2, 8-linked N-acetylneuraminate and is a virulence factor in this organism (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].

Locations: cytosol

Molecular Weight of Polypeptide: 48.736 kD (from nucleotide sequence), 50.0 kD (experimental) [Vann87 ]

Unification Links: Pride:P13266 , Protein Model Portal:P13266 , UniProt:P13266

Relationship Links: Entrez-Nucleotide:PART-OF:J05023 , InterPro:IN-FAMILY:IPR003329 , InterPro:IN-FAMILY:IPR013831 , Pfam:IN-FAMILY:PF02348

Gene-Reaction Schematic: ?

GO Terms:

Cellular Component: GO:0005829 - cytosol [Vann87]

Credits:
Created 04-Mar-2009 by Fulcher CA , SRI International


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

N-acetyl-β-neuraminate + CTP <=> CMP-N-acetyl-β-neuraminate + diphosphate

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.

In Pathways: superpathway of sialic acids and CMP-sialic acids biosynthesis , CMP-N-acetylneuraminate biosynthesis II (bacteria)

Summary:
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].

Cofactors or Prosthetic Groups: Mg2+ [Vann87], Mn2+ [Vann87]

Kinetic Parameters:

Substrate
Km (μM)
Citations
N-acetyl-β-neuraminate
4000.0
[Vann87]
CTP
310.0
[Vann87]

pH(opt): 9-10 [Vann87]


References

Angata02: Angata T, Varki A (2002). "Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective." Chem Rev 102(2);439-69. PMID: 11841250

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

Mizanur08a: Mizanur RM, Pohl NL (2008). "Bacterial CMP-sialic acid synthetases: production, properties, and applications." Appl Microbiol Biotechnol 80(5);757-65. PMID: 18716769

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

Zapata89: Zapata G, Vann WF, Aaronson W, Lewis MS, Moos M (1989). "Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene." J Biol Chem 264(25);14769-74. PMID: 2549035


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Sat Dec 20, 2014, biocyc11.