The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases

Konz, Dirk; Klens, Andrea; Schörgendorfer, Kurt; Marahiel, Mohamed A.

doi:10.1016/s1074-5521(97)90301-x

Cited by 211 publications

(213 citation statements)

References 49 publications

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“…First attempts to excise and express the EntF A domain as a typical 50-to 60-kDa A domain fragment failed to give soluble folded protein with any detectable activity (23), highlighting the ongoing unpredictability of stable folding of internal NRPS domains (26,(32)(33)(34). Therefore, we retained the native N terminus of EntF and expressed the two domain C-A fragment of EntF as a 108-kDa protein (residues 1-974), which proved to be soluble and contain an active A domain when purified as a C-terminal His 6 -tagged fragment.…”

Section: Discussionmentioning

confidence: 99%

“…ATP-pyrophosphate (PP i ) exchange was assayed as described previously (21). Reactions (100 L) contained 75 mM Tris⅐HCl (pH 7.5), 10 mM MgCl 2 , 1 mM tris-(2-carboxyethyl)phosphine (Molecular Probes), 1 mM sodium [ 32 Reactions for analysis by MS were prepared by using final concentrations of 50 M SrfB1 PCP, 5 M EntF 1-974-His 6 , and 10 mM unlabeled serine. Samples were separated by HPLC followed by electrospray MS, both performed by the Howard Hughes Medical Institute Biopolymers Facility at Harvard Medical School.…”

Section: Atp-[ 32 P]ppi Exchangementioning

confidence: 99%

“…The classic assay for A domains in both NRPS and aminoacyl-tRNA synthetases is the amino aciddependent exchange of radiolabel from 32 PP i into ATP (7), which measures reversible formation of aminoacyl-AMP, the first half reaction, and allows a clear measure of amino acid selectivity in this step. The assay is uninformative with regard to the second half reaction of A domains, the capture of the aminoacyl portion of the aminoacyl-AMP by the thiol of the Ppant prosthetic group of the paired PCP domain.…”

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confidence: 99%

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

Ehmann

Shaw-Reid

Losey

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

116

101

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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 acyl͞aminoacyl-AMP followed by adventitious thiolation by the heterologous PCPs in solution was addressed by measuring catalytic turnover of pyrophosphate (PPi) released from the adenylation domain. The holo SrfB1 PCP protein as well as Ybt PCP1 did not stimulate an increase in PPi 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. N onribosomal peptide synthetases (NRPS) activate a broad range of amino acids, including numerous nonproteinogenic amino acids (1, 2) and incorporate them into growing peptidyl chains as an elongating series of peptidyl-S-enzyme intermediates. This is an RNA-independent templated chaingrowth process with an assembly line organization of different catalytic and carrier protein domains whose placement and function determine the length and structure of the released peptide natural product. Both the amino acid monomers and the elongating peptide chains are tethered as thioester intermediates (acyl-S-enzymes) to the terminal thiol of phosphopantetheinyl (Ppant) moieties of peptidyl carrier protein (PCP) domains, also known as thiolation (T) domains for their functions as thioltethering way stations. The Ppant thiol in the PCP domains is added posttranslationally by priming enzymes (3, 4) to a conserved serine side chain in each PCP domain, converting it from inactive apo form to holo form capable of aminoacylation.The 8-to 10-kDa peptidyl chain-carrying PCP domains are interspersed among and paired with two catalytic domains that make up the core of each NRPS elongation module: 50-to 60-kDa adenylation (A) domains for each amino acid to be activated and 50-to 60-kDa condensation (C) domains for each peptide bond-forming chain-translocating step. Thus a typical NRPS module consists of the core triad of (C-A-PCP) n domains as the functional unit competent for: selection and activation of a given amino acid as the aminoacyl-AMP (Fig. 1, Step 1), covalent loading of the aminoacyl m...

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Section: Discussionmentioning

confidence: 99%

Section: Atp-[ 32 P]ppi Exchangementioning

confidence: 99%

mentioning

confidence: 99%

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

Ehmann

Shaw-Reid

Losey

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

116

101

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“…A large synthetase complex builds the peptide by connecting individual fragments. The genome sequence of this synthetase complex has recently been determined [5]. As a consequence of the non-standard pathway, bacitracin A exhibits some unusual features not commonly found in peptides.…”

Section: Introductionmentioning

confidence: 99%

EXAFS study of zinc coordination in bacitracin A

Drabløs

Nicholson

Rønning

1999

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Bacitracin is a dodecapeptide antibiotic produced by Bacillus sp. The antibacterial activity depends upon the peptide binding a divalent metal. Hitherto, the exact coordination of the cation has not been established. In particular the role played by the sulphur and nitrogen atoms of the thiazoline ring of bacitracin A has not been clear. Here the coordination of Zn 2 by bacitracin A has been studied using extended X-ray absorption fine structure. The experimental data are consistent with a model in which zinc is coordinated by one oxygen and three nitrogen atoms with the sulphur atom of the thiazoline ring not being directly involved in the zinc coordination. ß

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“…These peptides are synthesized by nonribosomal peptide synthases (7)(8)(9), and contain both D-and L-amino acids. The peptides are cyclized into lariat structures via condensation of the e-amino group of a lysine side chain with the peptide C terminus (10).…”

mentioning

confidence: 99%

High-resolution crystal structure reveals molecular details of target recognition by bacitracin

Economou

Simon

Loll

2013

Proc. Natl. Acad. Sci. U.S.A.

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Bacitracin is a metalloantibiotic agent that is widely used as a medicine and feed additive. It interferes with bacterial cell-wall biosynthesis by binding undecaprenyl-pyrophosphate, a lipid carrier that serves as a critical intermediate in cell wall production. Despite bacitracin’s broad use, the molecular details of its target recognition have not been elucidated. Here we report a crystal structure for the ternary complex of bacitracin A, zinc, and a geranyl-pyrophosphate ligand at a resolution of 1.1 Å. The antibiotic forms a compact structure that completely envelopes the ligand’s pyrophosphate group, together with flanking zinc and sodium ions. The complex adopts a highly amphipathic conformation that offers clues to antibiotic function in the context of bacterial membranes. Bacitracin’s efficient sequestration of its target represents a previously unseen mode for the recognition of lipid pyrophosphates, and suggests new directions for the design of next-generation antimicrobial agents.

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The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases

Abstract: The genes encoding the bacitracin synthetases BA1, BA2 and BA3 are organized in an operon, the structure of which reflects the modular architecture expected of peptide synthetases. In addition, a putative thiazoline ring formation domain was identified in the BA1 gene.

Cited by 211 publications

References 49 publications

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

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

EXAFS study of zinc coordination in bacitracin A

High-resolution crystal structure reveals molecular details of target recognition by bacitracin

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