Engineered Chimeras Unveil Swappable Modular Features of Fatty Acid and Polyketide Synthase Acyl Carrier Proteins
Yae In Cho, Claire Armstrong, Ariana Sulpizio, Kofi Acheampong, Kameron N. Banks, Oishi Bardhan, Sydney J. Churchill, Annie E. Connolly-Sporing, Callie E. W. Crawford, Peter Cruz Parrilla, Sarah M. Curtis, Lauren M. De La Ossa, Samuel C. Epstein, Clara Farrehi, Grayson S. Hamrick, William J. Hillegas, Austin Kang, Olivia C. Laxton, Joie Ling, Sara M. Matsumura, Victoria M. Merino, Shahla H. Mukhtar, Neel J. Shah, Casey H. Londergan, Clyde A. Daly, Bashkim Kokona, Louise K. Charkoudian
Abstract
FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based cross-linking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step toward the strategic engineering of "un-natural" natural products.