Natural products from bacteria are an important source for medical drugs, however, to fully benefit from them we need to understand their biosynthesis. The bacterial strain Actinomadura parvosata produces kistamicin, a cyclic peptide natural product with antiviral and weak antibacterial activity¹. It belongs to the glycopeptide antibiotics (GPAs) such as the most famous member vancomycin, which blocks bacterial cell wall maturation, although kistamicin does not function by interfering with the cell wall biosynthesis. The activity of GPAs is based on the characteristic three-dimensional conformation of the heptapeptide, which is due to the intramolecular crosslinks catalysed by a set of P450 enzymes. Nevertheless, kistamicin shows some clear differences to most GPAs, particularly, the nature of the crosslinks is changed and there are only two P450 enzymes in contrast to three crosslinks.

In this work, we have characterised the kistamicin crosslinking cascade by in vivo and in vitro turnover experiments. OxyC is a promiscuous biocatalyst for the insertion of multiple crosslinks into peptides containing phenolic amino acids. Furthermore, structural analysis and different interaction assays show that both Oxys function through interactions with the unique GPA X-domain². This work shows that, despite the differences in structure and activity of kistamicin when compared to the more common GPAs, the mechanism of peptide crosslinking remains conserved.³