Modular polyketide synthases (PKSs) are among the largest integrated enzyme complexes known, organizing clusters of catalytic domains into assembly lines which produce bioactive products that range from antibiotics to toxins. We are interested in how these systems use global conformational changes, protein-protein interfaces and protein-substrate recognition to control the growth of polyketide chains.
The key players in each module are acyl carrier proteins (ACPs), which serve as attachment points for the growing substrate chain and shuttle intermediates between each active site. Focusing on the erythromycin-producing synthase DEBS, we used NMR spectroscopy and isothermal calorimetry to demonstrate that processes in PKS systems can occur using fundamentally different mechanisms. For example, a specific protein-protein interface is required for phosphopantetheinyl transferase to charge ACP with a prosthetic group, but transfer of a polyketide chain from ACP to the terminal thioesterase domain is governed solely by recognition of the correct substrate chemistry. We have also determined solution structures for a series of novel “docking domains”, which regulate intermolecular substrate transfers in multi-subunit PKS and non-ribosomal peptide synthetase (NRPS) systems.
Taken together, these findings have important implications for attempts to engineer modified PKS and NRPS enzymes that make novel compounds.
Lab members: Mona Bassuni
1. Vance, S., Tkachenko, O., Thomas, B., Bassuni, M., Hong, H., Nietlispach, D., Broadhurst, W. (2016) Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase. Biochemical Journal 473, 1097-1110.
2. Murphy, A.C., Hong, H., Vance, S., Broadhurst, R.W., Leadlay, P.F. (2016) Broadening substrate specificity of a chain-extending ketosynthase through a single active site mutation. Chemical Communications 52, 8373-8376.
3. Ali M. & Broadhurst R.W. (2013) Solution structure of the QUA1 dimerization domain of pXqua, the Xenopus ortholog of Quaking. PLoS ONE 8, e57345.
4. Tran L., Broadhurst R.W., Tosin M., Cavalli A. & Weissman K.J. (2010) Insights into protein-protein and enzyme-substrate interactions in modular polyketide synthases. Chemistry & Biology 17, 705-716.
5. Richter C.D., Nietlispach D., Broadhurst R.W. & Weissman K.J. (2008) Multienzyme docking in hybrid megasynthases. Nature Chemical Biology 4, 75-81.
6. Broadhurst R.W., Nietlispach D., Wheatcroft M.P., Leadlay P.F., Weissman K.J. (2003) The structure of docking domains in modular polyketide synthases. Chemistry & Biology 10, 723-731.