Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin

Abstract

Lasso peptides are a new class of ribosomally synthesized and post-translationally modified peptides and thus far are only isolated from proteo-and actinobacterial sources. Typically, lasso peptide biosynthetic gene clusters encode enzymes for biosynthesis and export but not for tailoring. Here, we describe the isolation of the novel lasso peptide paeninodin from the firmicute Paenibacillus dendritiformis C454 and reveal within its biosynthetic cluster a gene encoding a kinase, which we have characterized as a member of a new class of lasso peptide-tailoring kinases. By employing a wide variety of peptide substrates, it was shown that this novel type of kinase specifically phosphorylates the C-terminal serine residue while ignoring those located elsewhere. These experiments also reveal that no other recognition motif is needed for efficient enzymatic phosphorylation of the C-terminal serine. Furthermore, through comparison with homologous HPr kinases and subsequent mutational analysis, we confirmed the essential catalytic residues. Our study reveals how lasso peptides are chemically diversified and sets the foundation for rational engineering of these intriguing natural products.

Dual substrate‐controlled kinase activity leads to polyphosphorylated lasso peptides

“…That the kinases, previously shown to be specific for the side chain of a C-terminal Ser, were able to transfer multiple phosphate groups to the truncated peptides indicates that all phosphate moieties were attached to a single Ser of each peptide. This indicates a high degree of substrate promiscuity, further compounding that observed previously [34], and is to the best of our knowledge unique among kinases.…”

“…The paeninodin gene cluster was mutated by SLIM using the plasmid pET41a-padeCA-thcoK-padeB1B2D [34] as template (see Table S3 for primers). To produce paeninodin variants, the modified gene cluster padeCA(DD22)-thcoKpadeB1B2D was expressed in M9 minimal medium [ (Table S4) were added after autoclaving] at 37°C for 3 days, as described for paeninodin [34].…”

“…To produce paeninodin variants, the modified gene cluster padeCA(DD22)-thcoKpadeB1B2D was expressed in M9 minimal medium [ (Table S4) were added after autoclaving] at 37°C for 3 days, as described for paeninodin [34]. Initially, 60 mL LB/kanamycin was inoculated with E. coli BL21(DE3) cells carrying pET41a-padeCA(DD22)-thcoK-padeB1B2D and grown overnight at 37°C.…”

“…Kinase-encoding operons were observed widely throughout firmicutes and proteobacteria, with gene arrangements clearly divided into two corresponding clades [30,34].…”

“…These results indicate that the kinases transferred multiple phosphate groups onto the native precursor peptides. However, S-ThcoA only contains two serine residues (SyanA contains eight), and we previously demonstrated strict specificity for the side chain of a C-terminal Ser by such kinases [34]. We thus hypothesized that the kinase first modifies the side chain of the C-terminal Ser and then sequentially transfers a phosphate group onto the existing modification.…”