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Multienzyme docking in hybrid megasynthetases

Abstract

Hybrid multienzyme systems composed of polyketide synthase (PKS) and nonribosomal polypeptide synthetase (NRPS) modules direct the biosynthesis of clinically valuable natural products in bacteria. The fidelity of this process depends on specific recognition between successive polypeptides in each assembly line—interactions that are mediated by terminal 'docking domains'. We have identified a new family of N-terminal docking domains, exemplified by TubCdd from the tubulysin system of Angiococcus disciformis An d48. TubCdd is homodimeric, which suggests that NRPS subunits in mixed systems self-associate to interact with partner PKS homodimers. The NMR structure of TubCdd reveals a new fold featuring an exposed β-hairpin that serves as the binding site for the C-terminal docking domain of the partner polypeptide. The pattern of charged residues on the contact surface of the β-hairpin is a key determinant of the interaction and seems to constitute a 'docking code' that can be used to alter binding affinity.

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Figure 1: Analysis of mixed PKS-NRPS systems.
Figure 2: NMR solution structure of the TubCdd dimer.
Figure 3: Binding of TubCdd and TubCdd mutants to immobilized TubBdd peptide, monitored by surface plasmon resonance.
Figure 4: Identification of the interaction interface on TubCdd by NMR.

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Acknowledgements

We thank M. Schaffer for guidance with the surface plasmon resonance experiments, and M. Hyvonen and A. Harrington for assistance with isothermal titration calorimetry. L. Foley (University of Cambridge) and R. Farndale (University of Cambridge) are acknowledged for synthesis of TubBdd peptide, and R. Müller (Saarland University) and A. Sandmann (Saarland University) for provision of cosmid F5. P. Leadlay is gratefully acknowledged for useful discussions and critical reading of this manuscript. An anonymous reviewer is thanked for helpful comments. C.D.R. was supported by a predoctoral studentship from the Boehringer Ingelheim Foundation and a fees-only studentship from the Medical Research Council. Funding to K.J.W. was provided by a Royal Society Dorothy Hodgkin Fellowship. Additional support was provided by grant 8/B18119 from the Biotechnology and Biological Sciences Research Council.

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C.D.R., experimental design, execution and analysis of all experiments relating to molecular biology, protein purification and biophysical characterization; also execution and analysis of NMR experiments and writing of the manuscript. D.N., design and execution of NMR experiments. R.W.B., design, execution and interpretation of NMR experiments, structure determination and writing of the manuscript. K.J.W., experimental concept, sequence and data analysis, and writing of the manuscript.

Corresponding authors

Correspondence to Carsten D Richter, R William Broadhurst or Kira J Weissman.

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Supplementary Figures 1–6, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 5576 kb)

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Richter, C., Nietlispach, D., Broadhurst, R. et al. Multienzyme docking in hybrid megasynthetases. Nat Chem Biol 4, 75–81 (2008). https://doi.org/10.1038/nchembio.2007.61

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