Understanding the crucial interactions between Cytochrome P450s and non-ribosomal peptide synthetases during glycopeptide antibiotic biosynthesis

Abstract

The importance of Cytochrome P450-catalyzed modifications of natural products produced by non-ribosomal peptide synthetase machineries is most apparent during glycopeptide antibiotic biosynthesis: specifically, the formation of essential amino acid side chains crosslinks in the peptide backbone of these clinically relevant antibiotics. These cyclization reactions take place whilst the peptide substrate remains bound to the non-ribosomal peptide synthetase in a process mediated by a conserved domain of previously unknown function-the X-domain. This review addresses recent advances in understanding P450 recruitment to non-ribosomal peptide synthetase-bound substrates and highlights the importance of both carrier proteins and the X-domain in different P450-catalyzed reactions.

A route to diastereomerically pure phenylglycine thioester peptides: crucial intermediates for investigating glycopeptide antibiotic biosynthesis

“…4,6,7 Fig. 1 GPA cyclisation in vitro is enabled by diastereomerically pure phenylglycine thioester peptides: teicoplanin seq.…”

“…6 In all these cases, however, the assessment of enzymatic activity is complicated by the necessity of peptide intermediates to be bound to peptidyl carrier protein (PCP)-domains, which serve as an attachment point for all amino acid and peptide intermediates during NRPS biosynthesis. 1,2,8 The ability to enzymatically load coenzyme A (CoA) substrates onto PCP domains using the promiscuous phosphopantetheinyl transferase Sfp has made a vital contribution to overcome this problem, as it allows biosynthetic steps to be interrogated without complete reconstitution of the NRPS.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…The most well-known compound class that exhibits multiple aromatic crosslinks that are installed by P450 enzymes are the glycopeptide antibiotics (GPAs), which include the representative examples vancomycin and teicoplanin. [240][241][242][243] The intramolecular cyclisation reactions catalysed by cytochrome P450 enzymes lead to a specic tertiary structure able to bind to the terminal D-Ala-D-Ala motif of the bacterial cell wall precursor lipid II and thereby blocking cell growth. As last-line antibiotics, they are the major treatment against multiresistant gram-positive pathogens like methicillin-resistant Staphylococcus aureus (MRSA).…”

“…[250][251][252] More recent work has shown that the process of P450 recruitment to the NRPS-bound peptide is more complicated than is the case for PCP-bound amino acids, with GPA biosynthesis relying on the X-domain, a conserved (albeit modied) condensation domain found between the PCP 253 and TE-domains in the nal NRPS module. 243,[254][255][256][257] The X-domain has an interaction interface that specically binds to the Oxy enzymes, with these continually binding to and releasing from the X-domain to ensure that complete crosslinking of the peptide occurs. 254 The X-domain is also required in the majority of cases to support efficient P450-catalysed crosslinking in vitro, with this requirement strictly enforced for OxyA and OxyE that act aer OxyB.…”

“…254 The X-domain is also required in the majority of cases to support efficient P450-catalysed crosslinking in vitro, with this requirement strictly enforced for OxyA and OxyE that act aer OxyB. 243,255,256,[258][259][260][261][262][263] Complestatin 264 and kistamicin 265 are structurally similar to GPAs, and also display several oxidative crosslinks whose generation has been ascribed to P450 enzymes, i.e. NRPS-bound modication with X-domain mediated P450 recruitment.…”