Recently through bioinformatics, it has been discovered that ribosomally synthesized and post-translationally modified peptides (RiPPs) often contain a precursor peptide recognition element that is homologous to PqqD, a protein involved in pyrroloquinoline quinone biosynthesis and also recently shown experimentally to bind its PqqA substrate. We investigated a PqqD homolog in lariatin biosynthesis. Lariatin is an anti-mycobacterial lasso peptide isolated from <i>Rhodococcus jostii</i> K01-B0171. Lasso peptide gene clusters have at a minimum, an A gene encoding the precursor, a B gene encoding the protease that removes the leader peptide of the precursor from the core peptide, and a C gene that encodes an enzyme involved in cyclization of the core peptide with the C-terminal end threaded through to form the final slipknot shape characteristic of lasso peptides. The B gene of many lasso peptide gene clusters encodes an N-terminal PqqD-like sequence followed by a cysteine protease domain. In the lariatin gene cluster these two proteins are split between two ORFs: LarB1 (PqqD homolog) and LarB2 (cysteine protease). Using bio-layer interferometry, we found that indeed LarB1 binds the leader peptide of the LarA precursor with submicromolar affinity. Guided by a homology model, we further characterized the interaction using unnatural amino acid (<i>p</i>-azido-phenylalanine) incorporation, photo-crosslinking and LC-MS/MS to determine which residues of LarB1 were in close proximity to LarA upon binding. Through these studies, we determined that LarB1 is a PqqD homolog that binds its precursor substrate in lariatin biosynthesis. This work provides new insights into lariatin biosynthesis and allows for the development of more potent lariatin variants.

PqqD homolog is a standalone leader peptide binding protein in lasso peptide biosynthesis

251st National Meeting (2016)

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