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Literature summary extracted from

  • Glaze, P.A.; Watson, D.C.; Young, N.M.; Tanner, M.E.
    Biosynthesis of CMP-N,N-diacetyllegionaminic acid from UDP-N,N-diacetylbacillosamine in Legionella pneumophila (2008), Biochemistry, 47, 3272-3282.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.5.1.101 gene lpg0752 or neuB, DNA and amino acid sequence determination and analysis, overexpression of the inactive C- and N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3), functional expression as MalE-NeuB fusion protein Legionella pneumophila
2.7.7.82 His-tagged protein expressed in Escherichia coli BL21(DE3) Legionella pneumophila
3.2.1.184 overexpressed in Escherichia coli as a His-tagged fusion protein Legionella pneumophila

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.184 0.0365
-
 UDP-N,N'-diacetylbacillosamine pH 7.5, 37°C Legionella pneumophila

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.5.1.101 additional information the synthase activity is dependent on the presence of a divalent metal ion Legionella pneumophila

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.5.1.101 2,4-diacetamido-2,4,6-trideoxy-alpha-D-mannopyranose + phosphoenolpyruvate + H2O Legionella pneumophila
-
 N,N'-diacetyllegionaminate + phosphate
-
 ?
2.5.1.101 2,4-diacetamido-2,4,6-trideoxy-alpha-D-mannopyranose + phosphoenolpyruvate + H2O Legionella pneumophila ATCC 33152D
-
 N,N'-diacetyllegionaminate + phosphate
-
 ?
2.5.1.101 additional information Legionella pneumophila the enzyme condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate, the reaction proceeded via a C-O bond cleavage process ?
-
 ?
2.5.1.101 additional information Legionella pneumophila ATCC 33152D the enzyme condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate, the reaction proceeded via a C-O bond cleavage process ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.5.1.101 Legionella pneumophila
-
 subsp. pneumophila, gene lpg0752 or neuB homologue, clustered with the neuC and neuA homologues
-
2.5.1.101 Legionella pneumophila ATCC 33152D
-
 subsp. pneumophila, gene lpg0752 or neuB homologue, clustered with the neuC and neuA homologues
-
2.7.7.82 Legionella pneumophila
-
-
-
2.7.7.82 Legionella pneumophila ATCC 33152D
-
-
-
3.2.1.184 Legionella pneumophila
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.5.1.101 recombinant C- and N-terminally and inactive His-tagged enzyme from Escherichia coli strain BL21(DE3) by metal chelating chromatography, recombinant active MalE-NeuB fusion protein by amylose affinity chromatography to high yields, the MalE protein is removed by thrombin treatment Legionella pneumophila
2.7.7.82 immobilized metal ion affinity chromatography (Ni2+) Legionella pneumophila
3.2.1.184 using Ni-NTA chromatography Legionella pneumophila

Reaction

EC Number Reaction Comment Organism Reaction ID
2.5.1.101 2,4-diacetamido-2,4,6-trideoxy-alpha-D-mannopyranose + phosphoenolpyruvate + H2O = N,N'-diacetyllegionaminate + phosphate C-O bond cleavage process reaction via a oxocarbenium ion intermediate generated by an initial attack of C-3 of phosphoenolpyruvate on the aldehyde of 2,4-diacetamido-2,4,6-trideoxymannose. This attack is facilitated by the divalent cation that serves as an electrophilic catalyst and polarizes the carbonyl of the aldehyde. Water then adds to the oxocarbenium ion intermediate to give a tetrahedral intermediate that subsequently collapses to generate phosphate and the product 2-oxo acid, overview Legionella pneumophila
a
3.2.1.184 UDP-N,N'-diacetylbacillosamine + H2O = UDP + 2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose the reaction proceeds with an inversion of stereochemistry at C-2 and retention of stereochemistry at C-1. Evidence of the cleavage of the C-1-O bond and the C-2-H bond supports an elimination/hydration mechanism similar to that of NeuC in sialic acid bisoynthesis Legionella pneumophila
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.5.1.101 2,4-diacetamido-2,4,6-trideoxy-alpha-D-mannopyranose + phosphoenolpyruvate + H2O
-
 Legionella pneumophila N,N'-diacetyllegionaminate + phosphate
-
 ?
2.5.1.101 2,4-diacetamido-2,4,6-trideoxy-alpha-D-mannopyranose + phosphoenolpyruvate + H2O
-
 Legionella pneumophila N,N'-diacetyllegionaminate + phosphate NMR spectroscopic product identification ?
2.5.1.101 2,4-diacetamido-2,4,6-trideoxy-alpha-D-mannopyranose + phosphoenolpyruvate + H2O
-
 Legionella pneumophila ATCC 33152D N,N'-diacetyllegionaminate + phosphate
-
 ?
2.5.1.101 2,4-diacetamido-2,4,6-trideoxy-alpha-D-mannopyranose + phosphoenolpyruvate + H2O
-
 Legionella pneumophila ATCC 33152D N,N'-diacetyllegionaminate + phosphate NMR spectroscopic product identification ?
2.5.1.101 additional information the enzyme condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate, the reaction proceeded via a C-O bond cleavage process Legionella pneumophila ?
-
 ?
2.5.1.101 additional information the enzyme does not show measurable activity with ManNAc Legionella pneumophila ?
-
 ?
2.5.1.101 additional information the enzyme condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate, the reaction proceeded via a C-O bond cleavage process Legionella pneumophila ATCC 33152D ?
-
 ?
2.5.1.101 additional information the enzyme does not show measurable activity with ManNAc Legionella pneumophila ATCC 33152D ?
-
 ?
2.7.7.82 CTP + N,N'-diacetyllegionaminic acid
-
 Legionella pneumophila CMP-N,N'-diacetyllegionaminic acid + diphosphate
-
 ?
2.7.7.82 CTP + N,N'-diacetyllegionaminic acid
-
 Legionella pneumophila ATCC 33152D CMP-N,N'-diacetyllegionaminic acid + diphosphate
-
 ?
3.2.1.184 additional information when UDP-GlcNAc is incubated with the enzyme, no product formation is observed Legionella pneumophila ?
-
 ?
3.2.1.184 UDP-N,N'-diacetylbacillosamine + H2O
-
 Legionella pneumophila 2,4-diacetamido-2,4,6-trideoxymannose + UDP
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.5.1.101 N,N'-diacetyllegionaminic acid synthase
-
 Legionella pneumophila
2.5.1.101 NeuB
-
 Legionella pneumophila
2.5.1.101 NeuB homologue
-
 Legionella pneumophila
2.7.7.82 CMP-N,N'-diacetyllegionaminic acid synthetase
-
 Legionella pneumophila
2.7.7.82 neuA
-
 Legionella pneumophila
3.2.1.184 neuC
-
 Legionella pneumophila
3.2.1.184 UDP-N-acetylglucosamine 2-epimerase
-
 Legionella pneumophila

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.5.1.101 25
-
 assay at Legionella pneumophila
3.2.1.184 37
-
 assay at Legionella pneumophila

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.184 59.1
-
 UDP-N,N'-diacetylbacillosamine pH 7.5, 37°C Legionella pneumophila

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.5.1.101 7.4
-
 assay at Legionella pneumophila
3.2.1.184 7.5
-
 assay at Legionella pneumophila

General Information

EC Number General Information Comment Organism
2.5.1.101 evolution the gene encoding the enzyme shows homology to known sialic acid biosynthetic gene neuB Legionella pneumophila
2.5.1.101 metabolism the three enzymes, UDP-N,N'-diacetylbacillosamine 2-epimerase, N,N'-diacetyllegionaminic acid synthase, and CMP-N,N'-diacetyllegionaminic acid synthetase, constitute a pathway that converts a UDP-linked bacillosamine derivative into a CMP-linked legionaminic acid derivative, the activated form of legionaminic acid used in lipopolysaccharide biosynthesis, overview Legionella pneumophila