Publications

• Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate.

  Gehring A.M., Bradley K.A., Walsh C.T.

  In Escherichia coli, the siderophore molecule enterobactin is synthesized in response to iron deprivation by formation of an amide bond between 2,3-dihydroxybenzoate (2,3-DHB) and l-serine and formation of ester linkages between three such N-acylated serine residues. We show that EntB, previously ... >> More

  In Escherichia coli, the siderophore molecule enterobactin is synthesized in response to iron deprivation by formation of an amide bond between 2,3-dihydroxybenzoate (2,3-DHB) and l-serine and formation of ester linkages between three such N-acylated serine residues. We show that EntB, previously described as the isochorismate lyase required for production of 2,3-DHB, is a bifunctional protein that also serves as an aryl carrier protein (ArCP) with a role in enterobactin assembly. EntB is phosphopantetheinylated near the C terminus in a reaction catalyzed by EntD with a kcat of 5 min-1 and a Km for apo-EntB of 6.5 microM. This holo-EntB is then acylated with 2,3-DHB in a reaction catalyzed by EntE, previously described as the 2,3-DHB-AMP ligase, with a kcat of 100 min-1 and a Km of <<1 microM for holo-EntB. The N-terminal 187 amino acids of EntB (isochorismate lyase domain) are not needed for reaction of EntB with either EntD or EntE as demonstrated by the equivalent catalytic efficiencies of the full-length EntB (residues 1-285) and the C-terminal EntB ArCP domain (residues 188-285) as substrates for both EntD and EntE. << Less

  Biochemistry 36:8495-8503(1997) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• MbtH-like proteins as integral components of bacterial nonribosomal peptide synthetases.

  Felnagle E.A., Barkei J.J., Park H., Podevels A.M., McMahon M.D., Drott D.W., Thomas M.G.

  The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unkno ... >> More

  The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins copurify with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs. << Less

  Biochemistry 49:8815-8817(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Enterobactin synthetase-catalyzed formation of P(1),P(3)-diadenosine-5'-tetraphosphate.

  Sikora A.L., Cahill S.M., Blanchard J.S.

  The EntE enzyme, involved in the synthesis of the iron siderophore enterobactin, catalyzes the adenylation of 2,3-dihydroxybenzoic acid, followed by its transfer to the phosphopantetheine arm of holo-EntB, an aryl carrier protein. In the absence of EntB, EntE catalyzes the formation of Ap(4)A, a m ... >> More

  The EntE enzyme, involved in the synthesis of the iron siderophore enterobactin, catalyzes the adenylation of 2,3-dihydroxybenzoic acid, followed by its transfer to the phosphopantetheine arm of holo-EntB, an aryl carrier protein. In the absence of EntB, EntE catalyzes the formation of Ap(4)A, a molecule that is implicated in regulating cell division during oxidative stress. We propose that the expression of EntE during iron starvation produces Ap(4)A to slow growth until intracellular iron stores can be restored. << Less

  Biochemistry 48:10827-10829(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Kinetic and inhibition studies of dihydroxybenzoate-AMP ligase from Escherichia coli.

  Sikora A.L., Wilson D.J., Aldrich C.C., Blanchard J.S.

  Inhibition of siderophore biosynthetic pathways in pathogenic bacteria represents a promising strategy for antibacterial drug development. Escherichia coli synthesize and secrete the small molecule iron chelator siderophore, enterobactin, in response to intracellular iron depletion. Here we descri ... >> More

  Inhibition of siderophore biosynthetic pathways in pathogenic bacteria represents a promising strategy for antibacterial drug development. Escherichia coli synthesize and secrete the small molecule iron chelator siderophore, enterobactin, in response to intracellular iron depletion. Here we describe a detailed kinetic analysis of EntE, one of six enzymes in the enterobactin synthetase gene cluster. EntE catalyzes the ATP-dependent condensation of 2,3-dihydroxybenzoic acid (DHB) and phosphopantetheinylated EntB (holo-EntB) to form covalently arylated EntB, a product that is vital for the final assembly of enterobactin. Initial velocity studies show that EntE proceeds via a bi-uni-uni-bi ping-pong kinetic mechanism with a k(cat) equal to 2.8 s(-1) and K(m) values of 2.5, 430, and 2.9 microM for DHB, ATP, and holo-EntB-ArCP, respectively. Inhibition and direct binding experiments suggest that, during the first half-reaction (adenylation), DHB binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), phosphopantetheinylated EntB binds to the enzyme followed by the release of products, AMP and arylated EntB. Two hydrolytically stable adenylate analogues, 5'-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) and 5'-O-[N-(2,3-dihydroxybenzoyl)sulfamoyl]adenosine (DHB-AMS), are shown to act as slow-onset tight-binding inhibitors of the enzyme with (app)K(i) values of 0.9 and 3.8 nM, respectively. Direct binding experiments, via isothermal titration calorimetry, reveal low picomolar dissociation constants for both analogues with respect to EntE. The tight binding of Sal-AMS and DHB-AMS to EntE suggests that these compounds may be developed further as effective antibiotics targeted to this enzyme. << Less

  Biochemistry 49:3648-3657(2010) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates.

  Ehmann D.E., Shaw-Reid C.A., Losey H.C., Walsh C.T.

  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 ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 97:2509-2514(2000) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF.

  Gehring A.M., Mori I., Walsh C.T.

  The siderophore molecule enterobactin, a cyclic trimeric lactone of N-(2,3-dihydroxybenzoyl)serine, is synthesized and secreted by Escherichia coli in response to iron starvation. Here we report the first reconstitution of enterobactin synthetase activity from pure protein components: holo-EntB, E ... >> More

  The siderophore molecule enterobactin, a cyclic trimeric lactone of N-(2,3-dihydroxybenzoyl)serine, is synthesized and secreted by Escherichia coli in response to iron starvation. Here we report the first reconstitution of enterobactin synthetase activity from pure protein components: holo-EntB, EntE, and holo-EntF. Holo-EntB and holo-EntF were obtained by pretreatment of apo-EntB and apo-EntF with coenzyme A and EntD, thereby eliminating the requirement for EntD in the enterobactin synthetase. The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates with a turnover rate of 120-140 min-1. There is no evidence for a stable complex of the enterobactin synthetase components. Mutation of holo-EntF in the thioesterase domain at the putative active site serine residue (Ser1138 to Ala) eliminated enterobactin synthetase activity; however, the mutant holo-EntF retained the ability to adenylate serine and to autoacylate itself by thioester formation between serine and its attached phosphopantetheine cofactor. The mutant holo-EntF also appeared to slowly release N-(2, 3-dihydroxybenzoyl)serine. << Less

  Biochemistry 37:2648-2659(1998) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection.

  Lai J.R., Fischbach M.A., Liu D.R., Walsh C.T.

  Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is ... >> More

  Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2/helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine in a structural model, and two of these positions are in the predicted helix 3 region. Subsequent in vitro studies are consistent with the hypothesis that these residues form a surface on EntB required for interaction with EntF. These results suggest that helix 3 is a major recognition element in EntB-ArCP and demonstrate the utility of selection-based approaches for studying NRPS biosynthesis. << Less

  Proc. Natl. Acad. Sci. U.S.A. 103:5314-5319(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.