Organic synthesis of candidate PKAL-1 substrates to probe the nemamide biosynthetic pathway

Melisa Gonzalez, Rebecca Butcher, Rongrong Yu, Xiang Li, Elijah Abraham, Matthew Gordon

University of Florida

Nemamides are hybrid polyketide-nonribosomal peptides that are produced by the nematode Caenorhabditis elegans to promote larval survival during starvation. These natural products are biosynthesized in an assembly-line manner by the polyketide synthase PKS-1 and nonribosomal peptide synthetase NRPS-1. Nemamides represent the first example of these types of natural products produced in animals. Many additional trans-acting enzymes participate in nemamide biosynthesis. Among these, Polyketide-ACP-Ligase-1 (PKAL-1) likely adenylates a key polyketide intermediate from PKS-1 and loads it onto a carrier protein of NRPS-1. A small library of potential substrates was synthesized to examine the substrate preferences of PKAL-1 to confirm its role in the nemamide biosynthetic pathway. Candidate substrates included 3-amino-5-hydroxyoctadecanoic acid, 3-aminoundecanoic acid derivatives, 3-amino-fatty acyl-beta-alanine, and (E)-5-hydroxyoctadec-2-enoic acid. Results from MALDI-TOF loading and adenylation assays revealed that the enzyme does not accept substrates with a 3-amino group. However, preliminary analysis showed that the enzyme was able to load the (E)-5-hydroxyoctadec-2-enoic acid, without a preference for the configuration of the stereogenic center at position 5. Future directions will focus on testing the selectivity of PKAL-1 by producing fatty acids with distinct functionalities at position 3. The more complex nemamide side chain will also be synthesized and examined as a potential PKAL-1 substrate.