Structure, biosynthesis, and bioactivity of nostolysamides

A recent genome mining study identified class II lanthipeptides encoded in Nostoc punctiforme PCC73102 that contain acyl groups conjugated to Lys side chains. The structure and bioactivity of these peptides, named nostolysamides, were not determined. In this study, we heterologously produced the nostolysamides by co- expression of the NpuA precursor peptide with an N-terminal SUMO tag with the class II lanthipeptide synthetase NpuM in Escherichia coli. We structurally characterized the NpuA-derived product and established the position of the thioether crosslinks. All four lanthionine and methyllanthionine residues were shown to have the DL configuration by Marfeys analysis. Tandem mass spectrometry as well as mutagenesis studies indicate an N-terminal non-overlapping methyllanthionine ring and three overlapping rings at the C-terminus for which the most likely ring pattern is proposed. After removal of the leader peptide, the resulting lanthipeptide exhibits antifungal activity against Candida species as well as antimicrobial activity against gram positive bacteria by disrupting cell membranes. The antibacterial activity is shown not to involve lipid II, consistent with the observed antifungal activity because fungi do not contain this bacterial cell wall precursor. The biosynthetic gene cluster also encodes an acetyltransferase NpuN that transfers long chain acyl groups to the side chain of a Lys residue in position 1 of the precursor peptide. In vitro studies of NpuN shows relatively broad substrate specificity with NpuN conjugating various acyl groups from acyl-CoA substrates to Lys1 in the nostolysamides. The acylation did not appreciably change the antifungal and antimicrobial activity of nostolysamide showing that it is not required for these activities.