The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates.
Enterobactin, the tris-(N-(2,3-dihydroxybenzoyl)serine) trilactone siderophore of Escherichia coli, is synthesized by a three-protein (EntE, B, F) six-module nonribosomal peptide synthetase (NRPS). In this work, the 142-kDa four-domain protein EntF was bisected into two double-domain fragments: a 108-kDa condensation and adenylation construct, EntF C-A, and a 37-kDa peptidyl carrier protein (PCP) and thioesterase protein, EntF PCP-TE. The adenylation domain activity of EntF C-A formed seryl-AMP but lost the ability to transfer the seryl moiety to the cognate EntF PCP-TE in trans. Seryl transfer to heterologous PCP protein fragments, the SrfB1 PCP from surfactin synthetase and Ybt PCP1 from yersiniabactin synthetase, was observed at rates of 0.5 min(-1) and 0.01 min(-1), respectively. The possibility that these slow acylation rates reflected dissociation of acyl/aminoacyl-AMP followed by adventitious thiolation by the heterologous PCPs in solution was addressed by measuring catalytic turnover of pyrophosphate (PP(i)) released from the adenylation domain. The holo SrfB1 PCP protein as well as Ybt PCP1 did not stimulate an increase in PP(i) release from EntF C-A or EntE. In this light, aminoacylations in trans between A and PCP domain fragments of NRPS assembly lines must be subjected to kinetic scrutiny to determine whether transfer is truly between protein domains or results from slow aminoacyl-AMP release and subsequent nonenzymatic thiol capture.
Ehmann
D E
DE
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Shaw-Reid
C A
CA
Losey
H C
HC
Walsh
C T
CT
D000249:Adenosine Monophosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D007700:Kinetics; D008025:Ligases / Q000737:chemistry*; D055666:Lipopeptides; D009097:Multienzyme Complexes / Q000737:chemistry*; D010453:Peptide Synthases / Q000737:chemistry*; D010456:Peptides, Cyclic; D010636:Phenols; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D017262:Siderophores / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013844:Thiazoles
D001426:Bacterial Proteins; D011756:Diphosphates; D029968:Escherichia coli Proteins; D055666:Lipopeptides; D009097:Multienzyme Complexes; D010456:Peptides, Cyclic; D010636:Phenols; D011994:Recombinant Proteins; D017262:Siderophores; D013438:Sulfhydryl Compounds; D013844:Thiazoles; C104398:yersiniabactin; C009365:surfactin peptide; D000249:Adenosine Monophosphate; D008025:Ligases; C066234:enterobactin synthetase; D010453:Peptide Synthases; C026269:2,3-dihydroxybenzoate - serine ligase; C061746:2,3-dihydroxybenzoate-AMP ligase, E coli