Exploring the compatibility of phosphopantetheinyl transferases with acyl carrier proteins spanning type II polyketide synthase sequence space

Phosphopantetheinyl transferases (PPTases) play an essential role in primary and secondary metabolism. These enzymes facilitate the posttranslational activation of acyl carrier proteins (ACPs) central to the biosynthesis of fatty acids and polyketides. Modulation of ACP-PPTase interactions is a promising approach to both increase access to desired molecular outputs and disrupt mechanisms associated with disease progression. However, such an approach requires understanding the molecular principles that govern ACP-PPTase interactions across diverse synthases. Through a multiyear, course-based undergraduate research experience (CURE), 17 ACPs representing a range of putative type II polyketide synthases, from actinobacterial and nonactinobacterial phyla, were evaluated as substrates for three PPTases (AcpS, Sfp, and vulPPT). The observed PPTase compatibility, sequence-level analyses, and predictive structural modeling suggest that ACP selectivity is driven by amino acids surrounding the conserved, modified serine on the ACP. We propose that vulPPT and Sfp interactions with ACPs are driven primarily by hydrophobic contacts, whereas AcpS may favor ACPs that exhibit high net-negative charge density, as well as a broad electronegative surface distribution. Furthermore, we report a plausible, hitherto unreported hydrophobic interaction between vulPPT and a conserved ACP crease upstream of the invariant serine, which may facilitate docking. This work provides a catalog of compatible and incompatible ACP-PPTase partnerships, highlighting specific regions on the ACP and/or PPTase that show promise for future strategic engineering and inhibitor development efforts. One-Sentence Summary: Seventeen acyl carrier proteins from diverse type II polyketide synthases were evaluated for their compatibility with three phosphopantetheinyl transferases; results, along with sequence level-analyses and predictive structural modeling, reveal specific regions that can guide future strategic engineering efforts.