Abstract
Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds1,2. Type I thioesterases (TEI) catalyse the release step from the assembly line3 of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4′-phosphopantetheine (4′-PP) cofactor that is covalently attached to thiolation (T) domains4,5,6,7,8,9. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo-T domains of NRPS and PKS systems10,11,12. Mispriming of 4′-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4′-PP cofactor, is acetylated in bacteria13. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes3,14 shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.
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Acknowledgements
We thank M. Strieker for editing the manuscript and B. Schaefer for her help and support in sample preparation. We thank Chi Scientific Inc. for the fast and high quality supply of the substrate peptides. The research was funded by the research grant BE-19/11 of the Deutsche Forschungsgemeinschaft (F.B. and M.A.M.); an enclosed fellowship (A.K.); the Centre for Biomolecular Magnetic Resonance at the University Frankfurt; the Cluster of Excellence Frankfurt (Macromolecular Complexes); and the Volkswagen Foundation (P.G.). A.K. thanks the Human Frontier Science Program Organization for a long-term fellowship awarded in April 2007.
Author Contributions A.K., V.D., F.B. and M.A.M. designed the experiments and defined the research for the SrfTEII. A.K., V.D. and C.T.W. wrote the manuscript and designed the experiments for the complex structure. A.K., F.B. and F.L. conducted the research including protein expression, data acquisition, resonance assignment and structure calculation. V.R.R. helped with resonance assignment and structure calculation of the SrfTEII. P.G. provided the volumes of the cavities of SrfTEI and of the complex SrfTEII–TycC3 T domain and supported the complex structure calculation. D.P.F. and G.W. recorded the NMR spectra of the protein complex. A.K. calculated the structure of the isolated SrfTEII, the structure of the protein complex (SrfTEII and TycC3–PCP) and performed all titration experiments. M.A.M. and M.R.M. provided the expression vectors for SrfTEII and the TycC3 T domain. M.A.M. provided the purification protocol and biochemical characterization of the SrfTEII. E.R.S. provided the non-hydrolysable CoA derivate and synthesized the peptide and amino acid modified substrate analogues.
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Koglin, A., Löhr, F., Bernhard, F. et al. Structural basis for the selectivity of the external thioesterase of the surfactin synthetase. Nature 454, 907–911 (2008). https://doi.org/10.1038/nature07161
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DOI: https://doi.org/10.1038/nature07161
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