Cloned (Comment) | Organism |
---|---|
gene ambE, encoded in the amb gene cluster, genetic organization, recombinant overexpression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)/pLys | Pseudomonas aeruginosa |
Protein Variants | Comment | Organism |
---|---|---|
D644A | site-directed mutagenesis, amino acid alterations in the A domain abolish loading of L-Glu entirely | Pseudomonas aeruginosa |
D644A/K1230T/S1985A | site-directed mutagenesis, the mutations abolish loading of L-Glu completely | Pseudomonas aeruginosa |
K1230T | site-directed mutagenesis, amino acid alterations in the A domain abolish loading of L-Glu entirely | Pseudomonas aeruginosa |
S1286A | site-directed mutagenesis, the mutation abolishes loading of L-Glu almost completely | Pseudomonas aeruginosa |
S1819A | site-directed mutagenesis, mutation of S1819 does not interfere with loading of L-Glu | Pseudomonas aeruginosa |
S1958A | site-directed mutagenesis, mutation of the active site Ser (S1958) of the TE domain does not interfere with loading of L-Glu | Pseudomonas aeruginosa |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Pseudomonas aeruginosa |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa | - |
AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa ATCC 15692 | - |
AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa 1C | - |
AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa PRS 101 | - |
AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa DSM 22644 | - |
AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa CIP 104116 | - |
AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa LMG 12228 | - |
AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa JCM 14847 | - |
AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate | Pseudomonas aeruginosa | - |
diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | Pseudomonas aeruginosa ATCC 15692 | - |
diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | Pseudomonas aeruginosa 1C | - |
diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | Pseudomonas aeruginosa PRS 101 | - |
diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | Pseudomonas aeruginosa DSM 22644 | - |
diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | Pseudomonas aeruginosa CIP 104116 | - |
diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | Pseudomonas aeruginosa LMG 12228 | - |
diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | Pseudomonas aeruginosa JCM 14847 | - |
diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa | - |
AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa ATCC 15692 | - |
AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa 1C | - |
AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa PRS 101 | - |
AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa DSM 22644 | - |
AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa CIP 104116 | - |
AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa LMG 12228 | - |
AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | Pseudomonas aeruginosa JCM 14847 | - |
AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas aeruginosa | Q9I1H3 | - |
- |
Pseudomonas aeruginosa 1C | Q9I1H3 | - |
- |
Pseudomonas aeruginosa ATCC 15692 | Q9I1H3 | - |
- |
Pseudomonas aeruginosa CIP 104116 | Q9I1H3 | - |
- |
Pseudomonas aeruginosa DSM 22644 | Q9I1H3 | - |
- |
Pseudomonas aeruginosa JCM 14847 | Q9I1H3 | - |
- |
Pseudomonas aeruginosa LMG 12228 | Q9I1H3 | - |
- |
Pseudomonas aeruginosa PRS 101 | Q9I1H3 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)/pLys by nickel affinity chromatography | Pseudomonas aeruginosa |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa | AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa ATCC 15692 | AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa 1C | AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa PRS 101 | AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa DSM 22644 | AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa CIP 104116 | AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa LMG 12228 | AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
(L-glutamyl)adenylate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa JCM 14847 | AMP + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate | - |
Pseudomonas aeruginosa | diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | - |
Pseudomonas aeruginosa ATCC 15692 | diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | - |
Pseudomonas aeruginosa 1C | diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | - |
Pseudomonas aeruginosa PRS 101 | diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | - |
Pseudomonas aeruginosa DSM 22644 | diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | - |
Pseudomonas aeruginosa CIP 104116 | diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | - |
Pseudomonas aeruginosa LMG 12228 | diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate | - |
Pseudomonas aeruginosa JCM 14847 | diphosphate + (L-glutamyl)adenylate | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa | AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa ATCC 15692 | AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa 1C | AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa PRS 101 | AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa DSM 22644 | AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa CIP 104116 | AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa LMG 12228 | AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
ATP + L-glutamate + holo-[L-glutamyl-carrier protein] | - |
Pseudomonas aeruginosa JCM 14847 | AMP + diphosphate + L-glutamyl-[L-glutamyl-carrier protein] | - |
ir | |
additional information | identification of L-Glu as the amino acid substrate activated by AmbE and loaded onto its T1 domain. The enzyme is also active with L-alanine instead of L-glutamate, but only in presence of the L-alanine-[L-alanyl-carrier protein] ligase AmbB (cf. EC 6.2.1.67), AmbB-dependent loading of L-Ala onto the T2 domain of AmbE, overview. Analysis of enzyme peptide fragment binding with substrate, peptide analysis by mass spectrometry, overview | Pseudomonas aeruginosa | ? | - |
- |
|
additional information | identification of L-Glu as the amino acid substrate activated by AmbE and loaded onto its T1 domain. The enzyme is also active with L-alanine instead of L-glutamate, but only in presence of the L-alanine-[L-alanyl-carrier protein] ligase AmbB (cf. EC 6.2.1.67), AmbB-dependent loading of L-Ala onto the T2 domain of AmbE, overview. Analysis of enzyme peptide fragment binding with substrate, peptide analysis by mass spectrometry, overview | Pseudomonas aeruginosa ATCC 15692 | ? | - |
- |
|
additional information | identification of L-Glu as the amino acid substrate activated by AmbE and loaded onto its T1 domain. The enzyme is also active with L-alanine instead of L-glutamate, but only in presence of the L-alanine-[L-alanyl-carrier protein] ligase AmbB (cf. EC 6.2.1.67), AmbB-dependent loading of L-Ala onto the T2 domain of AmbE, overview. Analysis of enzyme peptide fragment binding with substrate, peptide analysis by mass spectrometry, overview | Pseudomonas aeruginosa 1C | ? | - |
- |
|
additional information | identification of L-Glu as the amino acid substrate activated by AmbE and loaded onto its T1 domain. The enzyme is also active with L-alanine instead of L-glutamate, but only in presence of the L-alanine-[L-alanyl-carrier protein] ligase AmbB (cf. EC 6.2.1.67), AmbB-dependent loading of L-Ala onto the T2 domain of AmbE, overview. Analysis of enzyme peptide fragment binding with substrate, peptide analysis by mass spectrometry, overview | Pseudomonas aeruginosa PRS 101 | ? | - |
- |
|
additional information | identification of L-Glu as the amino acid substrate activated by AmbE and loaded onto its T1 domain. The enzyme is also active with L-alanine instead of L-glutamate, but only in presence of the L-alanine-[L-alanyl-carrier protein] ligase AmbB (cf. EC 6.2.1.67), AmbB-dependent loading of L-Ala onto the T2 domain of AmbE, overview. Analysis of enzyme peptide fragment binding with substrate, peptide analysis by mass spectrometry, overview | Pseudomonas aeruginosa DSM 22644 | ? | - |
- |
|
additional information | identification of L-Glu as the amino acid substrate activated by AmbE and loaded onto its T1 domain. The enzyme is also active with L-alanine instead of L-glutamate, but only in presence of the L-alanine-[L-alanyl-carrier protein] ligase AmbB (cf. EC 6.2.1.67), AmbB-dependent loading of L-Ala onto the T2 domain of AmbE, overview. Analysis of enzyme peptide fragment binding with substrate, peptide analysis by mass spectrometry, overview | Pseudomonas aeruginosa CIP 104116 | ? | - |
- |
|
additional information | identification of L-Glu as the amino acid substrate activated by AmbE and loaded onto its T1 domain. The enzyme is also active with L-alanine instead of L-glutamate, but only in presence of the L-alanine-[L-alanyl-carrier protein] ligase AmbB (cf. EC 6.2.1.67), AmbB-dependent loading of L-Ala onto the T2 domain of AmbE, overview. Analysis of enzyme peptide fragment binding with substrate, peptide analysis by mass spectrometry, overview | Pseudomonas aeruginosa LMG 12228 | ? | - |
- |
|
additional information | identification of L-Glu as the amino acid substrate activated by AmbE and loaded onto its T1 domain. The enzyme is also active with L-alanine instead of L-glutamate, but only in presence of the L-alanine-[L-alanyl-carrier protein] ligase AmbB (cf. EC 6.2.1.67), AmbB-dependent loading of L-Ala onto the T2 domain of AmbE, overview. Analysis of enzyme peptide fragment binding with substrate, peptide analysis by mass spectrometry, overview | Pseudomonas aeruginosa JCM 14847 | ? | - |
- |
Subunits | Comment | Organism |
---|---|---|
More | modular structure of AmbE with domains for adenylation, thiolation, condensation, methylation, and thioester cleavage. AmbE may have an additional domain of unknown function at its N-terminus and the C domain is atypical, domain architecture, overview | Pseudomonas aeruginosa |
Synonyms | Comment | Organism |
---|---|---|
ambE | - |
Pseudomonas aeruginosa |
More | see also EC 6.2.1.67 | Pseudomonas aeruginosa |
non-ribosomal peptide synthetase | - |
Pseudomonas aeruginosa |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
28 | - |
assay at | Pseudomonas aeruginosa |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Pseudomonas aeruginosa |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
4'-phosphopantetheine | phosphopantetheinylation of recombinant apoenzyme AmbE by a purified promiscuous phosphopantetheinyl transferase Sfp at pH 7.5, 28°C | Pseudomonas aeruginosa | |
ATP | - |
Pseudomonas aeruginosa |
General Information | Comment | Organism |
---|---|---|
metabolism | Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB)is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE, EC 6.2.1.67 and EC 6.2.1.68, respectively), and two iron(II)/alpha-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. The AmbB substrate is identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE are loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurs only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE result in the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD are included in the assay, these peptides are no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide is found at T2, importance of flanking L-Ala residues in the precursor tripeptide | Pseudomonas aeruginosa |
additional information | enzyme AmbE presents the typical modular structure of non-ribosomal peptide synthetases (NRPSs) | Pseudomonas aeruginosa |
physiological function | the enzyme is involved in biosynthesis of Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB), which proceeds via a precursor tripeptide. Identification of the building blocks of AMB biosynthesis and modelling, assembly of a tripeptide AmbB precursor on AmbE, overview | Pseudomonas aeruginosa |