Characterizing the Biochemical Role of an Unusual Fatty Acyl-AMP Ligase from a Hybrid PKS-NRPS Pathway in Nematodes

PKAL-1 is a fatty acyl-AMP ligase that plays an essential role in the biosynthesis of the nemamides, hybrid polyketide-nonribosomal peptides from nematodes that promote recovery from starvation-induced larval arrest. This enzyme traffics biosynthetic intermediates between two enzymatic assembly lines: the hybrid polyketide synthase-nonribosomal peptide synthetase PKS-1 and the nonribosomal peptide synthetase NRPS-1. Specifically, a <i>pkal-1</i> mutant worm strain does not produce the nemamides but accumulates a β-amino-containing polyketide, named nematide A, which is made by PKS-1 and is potentially loaded by PKAL-1 onto NRPS-1. Here, however, we show that PKAL-1 does not accept β-amino-containing substrates. Furthermore, phylogenetic and structural analyses of PKAL-1 indicate that it is likely missing key residues in its active site for binding to a β-amino group. By analyzing a panel of alternative substrates in biochemical assays, we show that PKAL-1 prefers α,β-unsaturated substrates. Our work potentially suggests an alternative model for nemamide biosynthesis whereby PKAL-1 loads an α,β-unsaturated substrate onto NRPS-1 and the β-amino group is installed later in the biosynthetic pathway. Furthermore, our data could indicate that nematide A is separate from the biosynthetic intermediates that are passed between PKS-1 and NRPS-1 in the construction of the nemamides and may have its own signaling function. Thus, PKAL-1 may help to regulate what is made by the PKS-1-NRPS-1 assembly line, either nematide A or the nemamides.