More than just recruitment: the X-domain influences catalysis of the first phenolic coupling reaction in A47934 biosynthesis by Cytochrome P450 StaH

Abstract

Glycopeptide antibiotic biosynthesis involves a complex cascade of reactions centred on a non-ribosomal peptide synthetase and modifiying proteins acting in trans, such as Cytochrome P450 enzymes. These P450s are responsible for cyclisation of the peptide via cross-linking aromatic amino acid side chains, which are a hallmark of the glycopeptide antibiotics. Here, we analysed the first cyclisation reaction in the biosynthesis of the glycopeptide antibiotic A47934. Our results demonstrate that the P450 StaH is recruited to the NRPS machinery through interaction with the X-domain present in the last A47934 NRPS module. We determined the crystal structure of StaH and showed that it is responsible for the first cyclisation in A47934 biosynthesis and additionally exhibits flexible substrate specificity. Our results further point out that the X-domain has an impact on the efficiency of the in vitro cyclisation reaction: hybrid PCP-X constructs obtained by domain exchange between A47934 and teicoplanin biosynthesis NRPS modules reveal that the X-domain from A47934 leads to decreased P450 activity and alternate stereochemical preference for the substrate peptide. We determined that a tight interaction between StaH and the A47934 X-domain correlates with decreased in vitro P450 activity: this highlights the need for glycopeptide antibiotic cyclisation to be a dynamic system, with an overly tight interaction interfering with substrate turnover in vitro.

Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934

“…This corresponds to the absorption spectra characteristic for P450s in the low-spin state, indicating the heme moiety of StaF to be present in its water-bound ferric form. Equivalent spectra were observed for related P450s such as StaH, OxyA tei , OxyB tei and OxyE tei , which are also involved in GPA cyclisation reactions [12,14,19,22]. Reduction of StaF by addition of sodium dithionite led to conversion of ferric to ferrous heme, which was accompanied by shift of the Soret maximum to λ = 422 nm and of the β/α bands to λ = 532 and 559 nm in the UV–visible spectrum.…”

“…Later diversification of the completely crosslinked peptide aglycones is the major source of diversity in natural GPAs, and occurs against the completed peptide aglycones [1,10–11]. The installation of the crosslinks has received significant attention using both in vitro [12–26] and in vivo [27–32] techniques, largely due to the synthetic challenge that these modifications represent. In vivo studies initially confirmed that the cytochrome P450s, known as the Oxy enzymes, are each responsible for the installation of a single ring in the GPA aglycones and that there is a conserved order of activity in both type-I and type-IV GPAs.…”

“…1) have revealed that the X-domain is in fact far from an innocent bystander during peptide oxidation and that switching this domain to other homologues can affect the selectivity of the Oxy enzymes for their peptide substrates [12]. Combined with the fact that only a single OxyA enzyme has been successfully characterised to date [13–14 16–17], we resolved to make a detailed structural and biochemical analysis of the OxyA homologue from the A47934 system, named StaF, to investigate not only some of the mechanistic features of the OxyA reaction but also the role of the X-domain on the activity of this enzyme and to determine whether the recruitment domain can also affect peptide selectivity for later Oxy enzymes in the GPA cyclisation cascade.…”

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.…”

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

“…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.…”