Photoreactive Pantetheinyl Mimics Reveal Design Limitations for Crypto-Acyl Carrier Proteins

Polyketide synthases (PKS), fatty acid synthases (FAS), and non-ribosomal peptide synthetases (NRPS) span a variety of protein architectures and catalytic modalities. Common to all these biosynthetic modalities is the centrality of a phosphopantetheinylated carrier protein, which shuttles substrate either inter- or intramolecularly between discrete domains that catalyze enzymatic transformations within the assembly line, resulting in these biosynthetic classes being termed "thiotemplated." In all thiotemplated biosynthetic assembly lines, transient protein-protein interactions mediated by the carrier protein are essential for chain extension and catalysis. In recent years, synthetic analogs of phosphopantetheine (Ppant), known as "crypto-ACPs," have become powerful tools for probing certain protein-protein interactions in thiotemplated systems. These crypto-ACP probes are usually created through a chemoenzymatic process. Most existing crypto-ACP probes are designed around mechanism-based approaches, which are aimed at capturing specific protein interactions within these pathways. To develop a more versatile probe capable of capturing a broader range of transient protein-protein interactions, we designed and synthesized a series of diazirine-based phosphopantetheinylated mimics suitable for photocrosslinking using a commonly employed beta-alanine linkage for synthetic tractability. We then evaluated the efficiency of loading Escherichia coli AcpP with these diazirine probes as well as crosslinking with Escherichia coli FabD. Examination of both loading and crosslinking revealed that deviations from the structure of the natural substrate (4'-phosphopantetheine) of the phosphopantetheinyl (Ppant) arm of the carrier protein resulted in inefficient loading, leading to poor or no crosslinking. These results highlight the limitations of synthetic and structural modularity in crypto-ACP design and will assist future strategies for interrogating protein-protein interactions of this nature.