Structural characterization of drug-producing polyketide synthase multienzymes by electron microscopy, small-angle X-ray scattering and allied biophysical and synthetic chemistry approaches – PKS STRUCTURE

The mega-enzyme polyketide synthases (PKSs) of bacteria and fungi synthesize many life-saving polyketide medicines, including anti-cancer, bactericidal, anti-fungal and cholesterol-lowering compounds, as well as numerous chemicals used in agriculture and animal husbandry. These huge proteins are composed of a series of catalytic and carrier protein domains, which cooperate to generate molecules of high structural and stereochemical complexity – features which are intimately linked to the bioactivities of these secondary metabolites. Genetic engineering of PKSs is a promising approach in both academia and the biotechnology sector to obtain desirable analogues with improved properties and increased value. Such a strategy might be used to address, for example, our urgent need for novel chemotherapeutics and agents to tackle bacterial antibiotic resistance. However, this strategy remains hampered by our insufficient understanding of PKS structural biology. The major scientific challenge is to determine the three-dimensional architectures of these gigantic multienzymes, providing the key insights into inter-domain interactions that will underpin more efficient PKS re-engineering. To address this knowledge gap, our four-team international German/French collaboration (A. Kirschning and R. J. Cox groups, University of Hannover, Germany (Partners 1 and 2); K. J. Weissman group, University of Lorraine, France (Partner 3) and S. Spinelli/C. Cambillau group, University of Marseille, France (Partner 4)) proposes to characterize a range of intact PKS modules of varying domain composition from two model modular PKSs of bacteria (trans-AT type), as well as an intact fungal, iterative PKS by a two-tiered approach: initial analysis by small-angle X-ray scattering (SAXS), followed by detailed investigation of select constructs at higher resolution by electron microscopy (both negative-staining and cryo-EM). The target proteins will be investigated in their apo and holo (modified with the prosthetic group phosphopantetheine) forms, as well as in the presence of native intermediates and domain cross-linking agents prepared by chemical synthesis. The project thus depends on the close collaboration of four laboratories with highly complementary skills, in line with the DFG’s and ANR’s objective of fostering strong, international research programs. The major scientific result will be to reveal the overall architectures of multiple PKS multienzymes, as well as large-scale conformational changes related to the functional states of the proteins. The data we obtain will dramatically increase the efficacy of synthetic biology experiments aimed at re-engineering PKS machineries towards the generation of new compounds with therapeutic potential. This project will thus further our ultimate goal of bringing engineering-derived polyketide analogues into clinical use via collaborations with pharmaceutical and/or biotechnology companies.