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Literature summary for 6.2.1.68 extracted from

  • Murcia, N.; Lee, X.; Waridel, P.; Maspoli, A.; Imker, H.; Chai, T.; Walsh, C.; Reimmann, C.
    The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor (2015), Front. Microbiol., 6, 170 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

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

Metals/Ions Comment Organism Structure
Mg2+ required Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

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

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 (Commentary)

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 and Products (Substrate)

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

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

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 [°C] Temperature Optimum Maximum [°C] Comment Organism
28
-
assay at Pseudomonas aeruginosa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Pseudomonas aeruginosa

Cofactor

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

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