Publications

• Dynamic thiolation-thioesterase structure of a non-ribosomal peptide synthetase.

  Frueh D.P., Arthanari H., Koglin A., Vosburg D.A., Bennett A.E., Walsh C.T., Wagner G.

  Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, inc ... >> More

  Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation-thioesterase (T-TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4'-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T-TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T-TE interaction described here provides a model for NRPS, PKS and FAS function in general as T-TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries. << Less

  Nature 454:903-906(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Molecular characterization of the Escherichia coli enterobactin cistron entF and coupled expression of entF and the fes gene.

  Pettis G.S., McIntosh M.A.

  The Escherichia coli entF gene, which encodes the serine-activating enzyme involved in enterobactin synthesis, has been localized to a 4.7-kilobase-pair DNA fragment inserted in the vector pBR328. This recombinant molecule, pITS32, restored the ability of an entF mutant to grow on low-iron medium ... >> More

  The Escherichia coli entF gene, which encodes the serine-activating enzyme involved in enterobactin synthesis, has been localized to a 4.7-kilobase-pair DNA fragment inserted in the vector pBR328. This recombinant molecule, pITS32, restored the ability of an entF mutant to grow on low-iron medium and to produce enterobactin. Examination of its translation products by minicell and electrophoretic analyses revealed a protein of approximately 160,000 daltons, which we identified as the EntF protein. A small DNA segment from pITS32 containing the translational start site for entF allowed the low constitutive expression of beta-galactosidase when cloned (pITS301) upstream of the lacZ structural gene in the vector pMC1403. In contrast, a clone (pITS312) containing the identical entF-lacZ fusion and a larger region upstream of entF including the entire fes gene and extending into the fepA gene (whose transcription is in the opposite direction relative to entF) expressed beta-galactosidase in high yet inducible amounts in response to fluctuations in the metabolic iron concentration. Transposon insertion mutations in the fes gene but not an insertion near the 5' region of fepA in pITS312 reduced this high inducible expression to the low constitutive level seen for pITS301. These observations are most readily explained by the presence of a regulatory region located upstream of fes which mediates the iron-regulated expression of a transcript that includes the fes and entF genes. << Less

  J. Bacteriol. 169:4154-4162(1987) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units.

  Chan Y.A., Boyne M.T. II, Podevels A.M., Klimowicz A.K., Handelsman J., Kelleher N.L., Thomas M.G.

  Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structu ... >> More

  Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis. << Less

  Proc. Natl. Acad. Sci. U.S.A. 103:14349-14354(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• 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.

• Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine.

  Rusnak F., Sakaitani M., Drueckhammer D., Reichert J., Walsh C.T.

  The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have ... >> More

  The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-[32P]PPi exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract. A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF. In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor. << Less

  Biochemistry 30:2916-2927(1991) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of EntF as a serine-activating enzyme.

  Reichert J., Sakaitani M., Walsh C.T.

  EntF is the enzyme responsible for serine activation during the biosynthesis of enterobactin (a cyclic trimer of N-dihydroxybenzoyl serine) in Escherichia coli. EntF has been overexpressed and purified to > 90% homogeneity. The enzyme has been shown to complement the entF-MK1 strain in the synthes ... >> More

  EntF is the enzyme responsible for serine activation during the biosynthesis of enterobactin (a cyclic trimer of N-dihydroxybenzoyl serine) in Escherichia coli. EntF has been overexpressed and purified to > 90% homogeneity. The enzyme has been shown to complement the entF-MK1 strain in the synthesis of 2,3-dihydroxybenzoyl serine derivatives and exhibits L-serine-dependent ATP[32P] pyrophosphate exchange activity with a Km for serine of 260 mM and a turnover number of 760 min-1. Release of PPi during incubation of EntF with serine and ATP was observed, but with a low turnover number of 1.0 min-1. These results suggested the presence of an enzyme-bound intermediate, which has been shown by gel filtration analysis to be (L-serine)adenylate. << Less

  Protein Sci. 1:549-556(1992) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.