Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase

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Abstract

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties1. 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 orientations2. 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.

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Figure 1: Summary of enterobactin synthesis.
Figure 2: Structure of the EntF T–TE fragment.
Figure 3: Interaction with Sfp.
Figure 4: Interaction with the C domain.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates have been deposited in the Protein Data Bank under accession code 2ROQ.

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Acknowledgements

We thank M. Sastry and A. Marintchev for advice in sample preparation; Z. Zhou for providing the Ser48Ala plasmid; Z.-Y. Sun for participating in the development of new NMR techniques; and P. Selenko for comments on the manuscript. This work was supported by NIH grants GM47467, EB 002026, and GM066360 and a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund (D.A.V.). A.K. thanks the Human Frontier Science Program for a long-term fellowship awarded in April 2007.

Author Contributions D.P.F., G.W. and C.T.W. designed research and wrote the manuscript. D.P.F. conducted the research including protein expression, resonance assignment and structure calculation. H.A. prepared various samples and helped during the acquisition of NMR experiments. D.A.V. prepared various samples. A.K. helped during the structure calculation and prepared the C domains EntD and Sfp samples. A.E.B. participated in early stages of the project.

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Correspondence to Dominique P. Frueh or Christopher T. Walsh or Gerhard Wagner.

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Frueh, D., Arthanari, H., Koglin, A. et al. Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase. Nature 454, 903–906 (2008) doi:10.1038/nature07162

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