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

Abstract

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. 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. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. 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, 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, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.

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.…”

“…We then tested the level of cyclisation seen in these peptides using the proven enzymatic coupling of OxyB van and OxyA tei , 7,[13][14][15]24,25 with the peptides themselves loaded onto a PCP-X di-domain construct excised from the final module of the teicoplanin NRPS machinery. 7 The results of the Oxy-catalysed turnover of peptides 4-5 demonstrated how vital the L-configuration of the C-terminal residue is for effective enzymatic crosslinking. For the racemic peptide 4, monocyclisation was now seriously impaired by the presence of the incorrect peptide diastereomer, which reduced the levels of monocyclisation to below 20% (Table 1).…”

Multitasking of the piRNA Silencing Machinery: Targeting Transposable Elements and Foreign Genes in the Bdelloid Rotifer Adineta vaga

“…The CYP450 enzymes catalyze oxidative transformation, which contributes to a broad array of biological functions in living organisms, such as activation or inactivation of many endogenous and exogenous bioactive compounds, particularly xenobiotic detoxification, or even modification of nonribosomal peptides (Guengerich 1991;Haslinger et al 2015). CYP enzymes are found in all kingdoms and domains of life (metazoans, plants, fungi, protists, bacteria, and archaea) and even in a virus.…”

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

“…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.…”

“…[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.…”