X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis

Glycopeptide antibiotics are biosynthesized by non-ribosomal peptide synthetases, which contain a previously uncharacterized ‘X-domain’ now shown to recruit three cytochrome P450 oxygenases that are necessary for the antibiotics to achieve their final, active conformation. A key glycopeptide synthesis enzyme The glycopeptide antibiotics such as vancomycin and teicoplanin, highly effective against Gram-positive bacterial infections, are secondary metabolites biosynthesized by non-ribosomal peptide synthetases. In vivo synthesis is currently the only option available to produce these highly complex structures, so understanding the biosynthesis pathway is of great clinical importance. In this manuscript, the authors determine the structure of the previously uncharacterized 'X-domain' that is strictly conserved in the final module of all glycopeptide non-ribosomal peptide synthetases. The X-domain interacts with two cytochrome P450s, and the structures of the X-domain both isolated and in complex with the first P450 oxygenase protein from teicoplanin biosynthesis reveals the inactive nature of the X-domain as well as how oxygenase recruitment occurs. Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome 1 . The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin 2 , 3 . These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 . However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain 13 , 14 , a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains,