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 ribosome1. 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 teicoplanin2,3. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation4,5,6,7,8,9,10,11,12. 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-domain13,14, 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, 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.

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Figure 1: Structure of the teicoplanin aglycone and a schematic pathway of teicoplanin biosynthesis by non-ribosomal peptide synthesis.
Figure 2: Structures of the X-domain and the X-domain–OxyBtei complex.
Figure 3: Interaction of the X-domain with Oxy proteins.
Figure 4: In vitro activity of OxyBtei and its homologues.
Figure 5: Coupled in vitro activity of OxyAtei and OxyBtei.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Structures have been deposited in the Protein Data Bank under accession numbers 4TX2 and 4TX3.

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Acknowledgements

The authors thank A. Koch for assistance with protein expression; S. Bell for redox proteins; M. Gradl for assistance with mass spectrometry; M. Tarnawski and A. Meinhart for assistance with crystal harvesting and data processing; I. Schlichting and J. Wray for discussions; C. Roome for IT support; R. Süssmuth and A. Truman for sharing unpublished data. Diffraction data were collected at the Swiss Light Source, X10SA beamline, Paul Scherrer Institute, Villigen, Switzerland. We thank the Heidelberg team for data collection and the PXII staff for their support in setting up the beamline. M.J.C. is grateful to I. Schlichting for constant encouragement and to the Deutsche Forschungsgemeinschaft (Emmy−Noether Program, CR 392/1-1) for financial support.

Author information

M.J.C. designed the study. K.H., M.P. and E.M. performed the biochemical experiments. C.B. performed the chemical synthesis and compound characterization. M.P., K.H. and M.J.C. solved the structures and performed the analysis. M.J.C. wrote the manuscript together with contributions from K.H., M.P. and C.B.

Correspondence to Max J. Cryle.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Overview and graphical summary of this study.

a, b, Final stages of teicoplanin aglycone biosynthesis (a) and vancomycin-type aglycone biosynthesis (b). c, d, In vitro binding studies demonstrated that the minimal interaction interface of OxyA–C is the conserved X-domain from the final module of the teicoplanin non-ribosomal peptide synthetase (c) and that the OxyBtei and OxyAtei enzymes required for peptide maturation are only active against peptide-bound PCP7-substrates that also contain this X-domain (d). These results support the hypothesis that in vivo aglycone formation occurs through the interaction of Oxy enzymes with module seven of the teicoplanin non-ribosomal peptide synthetase that then act upon the NRPS-bound linear heptapeptide precursor.

Extended Data Figure 2 Interaction of Oxy enzymes with NRPS domains studied by analytical size-exclusion chromatography and multi-angle light scattering.

a, b, Profiles of OxyBtei (red) shown with the profiles of the corresponding NRPS domain constructs and variants (orange) and mixtures of the binding partners in a 1:3 ratio (blue). c, Profiles of OxyAtei, OxyCtei, OxyEtei (red) shown with the profiles of the corresponding GB1–X constructs (orange) and mixtures of the binding partners in a 1:3 ratio (blue). Solid lines represent detection at 415 nm and dashed lines at 280 nm detection.

Extended Data Figure 3 Interaction of Oxy enzymes with NRPS domains studied by native-PAGE.

a, Top, overview of electrophoretic migration behaviour of OxyBvan and OxyBtei in the absence/presence of different NRPS domain constructs (GB1–PCP7, –PCP7–X, –PCP7–X–TE, –X; 1:3 molar ratio). Bottom, interaction studies of OxyBtei with NRPScep proteins and OxyBvan with NRPStei proteins. b, Overview of electrophoretic migration behaviour of OxyAtei, Btei and Ctei in the absence/presence of GB1–Xtei or GB1–PCP7–X–TEtei (1:3). c, Titration of OxyBtei (10 µM) with GB1–Xtei (0 to 30 µM) leading to the appearance of a new band with low electrophoretic mobility and the disappearance of free OxyBtei at equimolar concentrations. The bands corresponding to an Oxy/X complex are marked with an asterisk, those corresponding to an Oxy/PCP7–X–TE complex with a triangle and those corresponding to Oxy/PCP7–X with a square.

Extended Data Figure 4 X-domain active site in comparison to TycC5–6.

a, b, Comparisons of the X-domain both in isolation (green/orange; a) and in complex with OxyBtei (green/orange; b) to the active site of TycC5–6 (grey, solved with a bound sulphate ion): the effect of the altered X-domain active-site motif is to block the active site. Selected residues are shown as sticks. Hydrogen bonds are indicated by dashed black/grey lines for the X-domain and in red for the TycC5–6/sulphate distance.

Extended Data Figure 5 Structure of OxyBtei and multiple sequence alignment of the Oxy enzymes.

a, Structure of OxyBtei determined in complex with the X-domain. Secondary structure elements are labelled and coloured grey with the following exceptions: B-helix and loop region (magenta), D-helix (cherry red), E-helix (blue), F-helix (orange), G-helix (yellow), I-helix (cyan), β1-sheet (green), β2-sheet (purple), β3-sheet (orange), β4-sheet (yellow), haem shown as sticks (C atoms, red; N atoms, blue; O atoms, orange) with the haem iron shown as a red sphere. b, Sequence alignment of Oxy proteins from Actinoplanes teichomyceticus teicoplanin gene cluster23,24. Secondary structure and colour scheme same as a; catalytic residues are shown in orange; residues important for X-domain interaction are indicated in the three boxed regions, are in bold and highlighted in yellow for OxyBtei. Colours used for the corresponding residues from OxyAtei, OxyCtei and OxyEtei indicate agreement with the consensus sequence (green, match; blue, comparable interaction; red, potential mismatch).

Extended Data Figure 6 Model of the PCP7–X–OxyBtei complex in two possible arrangements.

ad, Possible PCP position determined from the alignment of the P450sky–PCP7 complex trapped using a covalent azole inhibitor with the OxyBtei–X-domain complex (a, b) and the structure of the P450BioI–ACP complex (c, d). The X-domain is shown in grey, OxyBtei is shown in yellow, the haem of OxyBtei is shown in red sticks. a, b, The PCP7 (sky) is shown in magenta and the azole inhibitor covalently bound to the PCP is shown as pink sticks. c, d, The ACP is shown in blue and the phosphopantetheine-bound fatty acid is shown as blue sticks. The distance between the C terminus of the PCP/ACP and the N terminus of the X-domain is shown in a and c.

Extended Data Figure 7 HPLC-MS analysis of P450-catalysed peptide monocyclization.

ad, Representative HPLC-MS chromatograms of in vitro OxyBtei (a), StaH (b), OxyBvan (c), CepF (d) turnover reactions with heptapeptide Tei7(Hpg3,7) or Van7(Hpg7) bound to GB1–PCP7–X (left) and GB1–PCP7 (right). Ions corresponding to the singly charged, linear (hydrolysed m/z 1,088, methylhydrazide m/z 1,116) and crosslinked monocyclic (hydrolysed m/z 1,086, methylhydrazide m/z 1,114 ) teicoplanin-like peptide and the linear (hydrolysed m/z 1,017.5, methylhydrazide m/z 1,045.5 ) and crosslinked monocyclic (hydrolysed m/z 1,015.5, methylhydrazide m/z 1,043.5 ) vancomycin-like peptide recorded using single-ion monitoring (SIM) in negative mode. Major peaks for each mass represent diastereomers due to racemization of the C-terminal Hpg residue and two regioisomers derived from methylhydrazine cleavage; smaller peaks can be caused by overlapping mass signal detection with ions from lower molecular weight species; additionally, racemization of such crosslinked products has also been previously observed39.

Extended Data Figure 8 HPLC-MS analysis of OxyBtei-catalysed cyclization of peptides presented by mutant variants of PCP7–X.

ad, Representative HPLC-MS chromatograms of in vitro OxyBtei turnover reactions with substrate heptapeptide Tei7(Hpg3,7) bound to the different GB1–PCP7–X mutants (X1: R167A, R171A (a); X2: E290A, D291A (b); X3: E377A, R382A (c); X13: R167A, R171A, E290A, D291A, E377A, R382A (d)). Occurrence of ions corresponding to the singly charged, linear peptide (hydrolysed m/z 1,088, methylhydrazide m/z 1,116) and the crosslinked monocyclic product (hydrolysed m/z 1,086, methylhydrazide m/z 1,114) were recorded using SIM in negative mode. The origins of the multiple peaks observed for each mass are described in Extended Data Fig. 7.

Extended Data Figure 9 HPLC-MS analysis of P450-catalysed peptide bicyclization.

ad, Representative HPLC-MS chromatograms of coupled in vitro OxyBtei plus OxyAtei (a), OxyCtei (b), or OxyAtei/OxyCtei (c) turnover reactions on heptapeptide Tei7(Hpg3,7) bound to GB1–PCP7–X (a, left, b, c) and GB1–PCP7 (a, right); or OxyBtei alone with hexapeptide Tei6(Hpg3) bound to MBP–PCP6 (d). Occurrence of ions corresponding to the singly charged, linear peptide (hydrolysed m/z 1,088, methylhydrazide m/z 1,116 (a–c); hydrolysed m/z 939, methylhydrazide m/z 967 (d)), crosslinked monocyclic product (hydrolysed 1,086 m/z, methylhydrazide 1,114 m/z (a–c); hydrolysed m/z 937, methylhydrazide m/z 965 (d)), crosslinked bicyclic product (hydrolysed m/z 1,084, methylhydrazide m/z 1,112) and the crosslinked tricyclic product (hydrolysed m/z 1,082, methylhydrazide m/z 1,110) was recorded using SIM in negative mode. The origins of the multiple peaks observed for each mass are described in Extended Data Fig. 7.

Extended Data Table 1 Crystallographic data and refinement statistics for the isolated X-domain and the X-domain in complex with OxyBtei

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Haslinger, K., Peschke, M., Brieke, C. et al. X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis. Nature 521, 105–109 (2015). https://doi.org/10.1038/nature14141

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