Nature doi:10.1038/nature14141

Credit: NATURE

Glycopeptide antibiotics such as vancomycin and teicoplanin are produced by nonribosomal peptide synthetases (NRPSs), with the initial peptide backbones further cross-linked by cytochrome P450 enzymes to create the three-dimensional shape needed to bind the antibiotics' cellular target, lipid II. Prior work demonstrated that the compounds remain on the NRPS while the modifications are introduced, but the mechanism by which the P450s are recruited to the NRPS was unclear. Haslinger et al. took interest in an uncharacterized 'X' domain that is near the end of all glycopeptide antibiotic NRPSs and phylogenetically related to condensation (C) domains yet missing residues known to be crucial for C-domain activity. The authors' crystal structure confirmed a typical C-domain structure and provided more support that the domain has an alternate function, as the side chains of two mutated residues block the canonical cofactor binding site. The authors thus tested for protein-protein interactions between the teicoplanin OxyB and truncated fragments of the NRPS. Gel filtration and mobility shift assays demonstrated a strong (low mM) interaction dependent only on the X domain for both the teicoplanin and vancomycin pathways. A crystal structure of the complex identified the interaction site and confirmed that both the P450's reductase partner and the antibiotic carrier protein would be able to access the appropriate sites on the P450 for catalysis. The authors further tested whether the other P450s might be recruited by the same mechanism: OxyA and OxyC, which act after OxyB, did bind the X domain, though with lower affinities, suggesting that interactions with the appropriate substrate could be driving sequential activity. Finally, in vitro assays confirmed that the activity of OxyBs from four different clusters was substantially increased by the X domain. These findings improve our understanding of NRPS machines and offer new opportunities to exploit these oxygenases for extended in vitro cyclization towards antibiotic discovery.