Studying protein‐protein interactions in fatty acid and polyketide biosynthetic pathways via site‐specific vibrational spectroscopy

Abstract

By virtue of their structural diversity and striking potency, natural products have long been used as therapeutic agents. Natural products are biosynthesized by assemblies of enzymes encoded in gene clusters. Repurposing the natural product biosynthetic repertoire to assemble “unnatural” natural products would have profound implications for human health. Our current lack of understanding of how these biosynthetic enzymes operate at a molecular level, which is partially attributed to the lack of tools that can capture the weak and transient interactions of the enzyme assemblies, stymies such engineering endeavors. Our lab previously showed that labeling acyl carrier proteins, which are central hubs of natural product assemblies, with a thiocyanate (SCN) vibrational probe provides an innovative method to study the proteins' conformational dynamics and interactions with ketosynthases. In this work, we evaluate the general utility of the probe by assessing ACP‐SCN interactions with other partner enzymes. The SCN probe was loaded onto the 4′‐phosphopantetheine (Ppant) arm of ACPs involved in fatty acid and actinorhodin type II polyketide biosynthetic pathways, AcpP and actACP, respectively. Changes in the CN stretching frequency were monitored as the ACPs were titrated with partner enzymes, 3‐hydroxyacyl‐ACP dehydrase (FabA) and actinorhodin polyketide ketoreductase (actKR). Upon a productive interaction of the ACP with its respective partner enzyme, the CN absorption band shifts to lower wavenumbers, which could correlate to the probe accessing the partner enzyme's hydrophobic catalytic active site. In light of these findings, the SCN probe appears to report on a wide range of ACP‐partner protein interactions. As a result, the probe could be a useful tool for future engineering studies.Support or Funding InformationWe would like to acknowledge an NIH Award #CHE‐1652424 to Louise K. Charkoudian as a source of funding for this project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.