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Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme

Nature Chemical Biology volume 20, pages 382–391 (2024)Cite this article

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Abstract

d-Amino acid residues, found in countless peptides and natural products including ribosomally synthesized and post-translationally modified peptides (RiPPs), are critical for the bioactivity of several antibiotics and toxins. Recently, radical S-adenosyl-l-methionine (SAM) enzymes have emerged as the only biocatalysts capable of installing direct and irreversible epimerization in RiPPs. However, the mechanism underpinning this biochemical process is ill-understood and the structural basis for this post-translational modification remains unknown. Here we report an atomic-resolution crystal structure of a RiPP-modifying radical SAM enzyme in complex with its substrate properly positioned in the active site. Crystallographic snapshots, size-exclusion chromatography–small-angle x-ray scattering, electron paramagnetic resonance spectroscopy and biochemical analyses reveal how epimerizations are installed in RiPPs and support an unprecedented enzyme mechanism for peptide epimerization. Collectively, our study brings unique perspectives on how radical SAM enzymes interact with RiPPs and catalyze post-translational modifications in natural products.

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Fig. 1: Epipeptide biosynthesis and overall structure of the EpeE radical SAM epimerase.
Fig. 2: Structure of EpeE in interaction with a peptide substrate and SAH cofactor.
Fig. 3: Structures of C223A mutant in interaction with two peptide substrates and SAH cofactor.
Fig. 4: Amino acid network in the active site of the radical SAM enzyme EpeE.
Fig. 5: Substrate interaction and proposed mechanism for EpeE.

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Data availability

Atomic coordinates and structure factors for the reported crystal structures in this work have been deposited in the Protein Data Bank upon accession codes 8AI1, 8AI2, 8AI3, 8AI4, 8AI5 and 8AI6. SAXS data have been deposited in the Small Angle Scattering Biological Data Bank upon accession codes SASDRS7 and SASDRR7. The data for this study are available within the paper and its Supplementary Information. Source data are provided in this paper.

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Acknowledgements

This work was supported by the French National Research Agency (ANR; grants ANR-17-CE11-0014 and ANR-20-CE44-0005 to O.B.). The authors are grateful to the EPR facilities available at the French EPR network (IR CNRS 3443, now INFRANALYTICS, FR2054) and the Aix-Marseille University EPR center. We acknowledge SOLEIL (Saint-Aubin, France) for the provision of synchrotron radiation facilities, and we would like to thank the PROXIMA-1 and SWING staff for assistance in using the beamline.

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Author notes

  1. These authors contributed equally: Xavier Kubiak, Ivan Polsinelli, Leonard M. G. Chavas, Cameron D. Fyfe.

Authors and Affiliations

  1. Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, ChemSyBio, Jouy-en-Josas, France

    Xavier Kubiak, Ivan Polsinelli, Cameron D. Fyfe, Alain Guillot, Laura Fradale, Clémence Brewee, Olivier Berteau & Alhosna Benjdia

  2. Nagoya University, Nagoya, Japan

    Leonard M. G. Chavas

  3. Aix-Marseille Université, CNRS, BIP, IMM, IM2B, Marseille, France

    Stéphane Grimaldi & Guillaume Gerbaud

  4. Synchrotron SOLEIL, HelioBio Group, L’Orme des Merisiers, Saint-Aubin, France

    Aurélien Thureau & Pierre Legrand

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  1. Xavier Kubiak
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Contributions

A.B. and O.B. performed research design and funding acquisition. X.K., I.P., L.M.G.C., C.D.F., A.G., S.G., G.G., A.T., P.L., O.B. and A.B. performed research. X.K., I.P., L.F. and C.B. performed protein production. X.K., I.P., L.M.G.C., C.D.F., A.G., S.G., G.G., A.T., P.L., O.B. and A.B. analyzed data. O.B. and A.B. wrote the manuscript with contributions from co-authors.

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Correspondence to Olivier Berteau or Alhosna Benjdia.

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Extended data

Extended Data Fig. 1 Topology diagram of EpeE, radical SAM binding motifs and coordination of SAM in EpeE and representative members of the radical SAM enzyme superfamily.

a, EpeE topology diagram showing a truncated α5/β6 TIM barrel in the radical SAM domain and T-SPASM domain with a single AuxI cluster. The binding region is depicted in bold line. The position of the [4Fe-4S] clusters (yellow and orange spheres) and the coordinating cysteine residues (yellow) are indicated. b, Radical SAM cluster of EpeE in interaction with SAH. Residues from the conserved: GGE, GXIXGXXE, ribose, β6 and CX3CXΦC motifs are colored according to their domain in EpeE (radical SAM domain). The unusual His-20 residue from the CX3CXΦC motif is highlighted. Hydrogen-bonds are shown with black lines, radical SAM [4Fe-4S] cluster with yellow and orange sticks and SAH with green sticks. c, The CX3CXΦC motif of structurally characterized members of the radical SAM superfamily of enzymes. The Φ residue is: Tyr in AnSME (green sticks) and CteB (brown sticks); Phe in SkfB (yellow sticks) and SuiB (violet sticks); Met in NosL (white sticks). As for CteB, the side chain of the Φ amino acid coordinates both the adenine and the ribose moiety, but only EpeE makes two H-bonds with the N6 and N7 of the adenine moiety.

Extended Data Fig. 2 Interactions stabilizing the T-SPASM domain and the bridging region of EpeE.

a, Overall structure of EpeE highlighting the bridging region (red cartoon) between EpeE domains (radical SAM domain in light blue, T-SPASM in teal). b, The bridging region (red cartoon) makes extensive H-bonds (black dotted lines) with α3’ helix and α3’-α4’ loop (green cartoon) (left panel). The C-terminal helix α5’ is stabilized by α3’ helix through H-bond and hydrophobic interactions (right panel). c, Comparison between the twitch and SPASM-domains of representative radical SAM enzymes. Similarly to twitch radical SAM enzymes (SkfB & BtrN), EpeE coordinates a single [4Fe-4S] cluster, however, the bridging region extends toward the location of AuxII cluster found in SPASM enzymes (AnSME, CteB & SuiB). The C-terminal α5’ helix of EpeE, absent in twitch-domains, is displaced at the opposite side of the AuxI cluster compared to the C-terminal α6’ helix of SPASM-domain enzymes AnSME and CteB which lies against the α6 helix of the TIM barrel (missing in EpeE). Only the AuxI cluster, β1’-β2’ anti-parallel sheets and α2’ helix positions are conserved in all enzymes. The TIM barrel domain is colored in white. The Twitch/SPASM domains are colored by protein. The AuxI and II clusters are shown as yellow and orange sticks.

Extended Data Fig. 3 EPR, UV-visible and HYSCORE analysis of EpeE in absence or presence of SAM.

a, Temperature dependence of EPR spectra of dithionite-reduced reconstituted samples of wild-type EpeE in the absence (left panel) or in the presence (right panel) of a 5-fold stoichiometric excess of SAM. The microwave power was adjusted at each temperature to avoid saturation effects. Spectra have been amplitude-normalized. Number of accumulations: 4. The signal of the SAM-bound [4Fe-4S]+ cluster relaxes significantly faster than the one detected in the unbound form. Indeed, the former broadens at temperatures above 6 K and is no longer visible at 30 K and above (right panel) whereas the latter is still detected without significant broadening at 30 K (left panel). Such differences in the relaxation behavior of the two forms allowed us to reveal partial conversion between these forms upon addition of SAM. Indeed, a weak contribution of the unbound form is detected in the sample incubated with a 5-fold excess of SAM when measured at 30 K (right panel). b, Power saturation experiments of dithionite-reduced reconstituted samples of wild-type EpeE in the absence (upper panel) or in the presence (lower panel) of a 5-fold stoichiometric excess of SAM. Peak-to-peak amplitudes between features measured as indicated by arrows on left spectra are plotted against square root of microwave power in a log-log plot (blue filled circles). The dotted line represents the non-saturation regime for which the EPR amplitude is proportional to the square root of the microwave power. Other experimental conditions: temperature, 15 K (upper panel) or 6 K (lower panel), microwave power, 0.1 mW (left spectra), number of accumulations, 4. c, UV visible analysis of EpeE wild-type (upper panel) and A3-mutant (lower panel). Before (gray line) and after (black line) anaerobic FeS cluster reconstitution. d, X-band HYSCORE spectra of dithionite-reduced reconstituted samples of wild-type EpeE in the presence (upper panel) or in the absence (lower panel) of a 5-fold stoichiometric excess of SAM. Only the low frequency region is shown. Experimental conditions are given in the Methods section. The low frequency region of the HYSCORE spectrum of the anaerobically reduced and reconstituted wild-type enzyme in the presence of SAM displays a complex set of signals in both the (+, +) and (-, +) quadrants which can be unambiguously assigned to a hyperfine coupling to a 14N nucleus in the intermediate coupling regime for which the isotropic part of the hyperfine coupling constant aiso is nearly equal to twice the 14N Larmor frequency, that is νI(14N) ≈ 1.1 MHz (upper panel). These signals are absent in the corresponding HYSCORE spectrum of the enzyme prepared in the same conditions but without SAM (lower panel). Importantly, the 14N HYSCORE pattern of the EpeE radical SAM [4Fe-4S]+ cluster measured in the presence of SAM is remarkably similar to the one of RlmN in the presence of SAM for which direct SAM binding to the radical SAM cluster has been established both by HYSCORE spectroscopy70 and X-ray crystallography71. This contrasts with the situation observed in cryoreduced TsrM for which HYSCORE measurements performed in the presence of SAM did not show 14N signals that would be consistent with SAM binding72. Therefore, we assign the 14N nucleus coupled to the [4Fe-4S]+ cluster in EpeE to the amino group of SAM coordinated to the unique iron of the cluster in a manner similar to that demonstrated in RlmN.

Extended Data Fig. 4 LC-MS analysis of EpeE incubated with peptides 4, 5, 6 & 7.

Activity of EpeE with peptide 4 (a), 5 (b), 6 (c) and 7 (d) was assayed in deuterated buffer. LC-MS analysis of peptide at T0 (upper left panel) and after 90 min incubation under anaerobic conditions (lower left panel). Comparison between the mass spectrum of the substrate (upper middle panel) and the product (lower middle panel) showed a + 1 Da mass increment, consistent with 2H-atom incorporation while mass spectrum analysis of the 5’-dAH is shown in right panel.

Extended Data Fig. 5 LC-MS/MS analysis of the peptides 4, 5, 6 and 7 and the reaction products formed after incubation with EpeE.

a, Mass fragmentation spectrum of peptide 4 (upper panel) and the epimerized peptide product (lower panel) (see Supplementary Tables 1 and 2 for full assignment). b, Mass fragmentation spectrum of peptide 5 (upper panel) and the epimerized peptide product (lower panel) (see Supplementary Tables 3 and 4 for full assignment). c, Mass fragmentation spectrum of peptide 6 (upper panel) and the epimerized peptide product (lower panel) (see Supplementary Tables 5 and 6 for full assignment). d, Mass fragmentation spectrum of peptide 7 (upper panel) and the epimerized peptide product (lower panel). (see Supplementary Tables 7 and 8 for full assignment). The relevant ions with a mass shift of +1 Da due to 2H incorporation after reaction with EpeE are highlighted.

Extended Data Fig. 6 Comparison between substrate-free and peptide-bound structures of EpeE.

a, Superimposition of substrate-free and peptide-bound structures of EpeE. b, Close-up view showing the major structural movements including the α3’-α5’ helices (indicated by arrows in panel a) of the T-SPASM domain. The substrate-free EpeE structure is shown in gray and the peptide-bound EpeE structure in pale cyan (chain A) and deep teal (chain B). Alignment of the substrate-free and -bound structures using all domains (634 residues) has a r.m.s.d. of 0.78 Å, as calculated using Coot SSM.

Extended Data Fig. 7 Peptide 5 bound in the active-site of wild-type EpeE structure.

a, The peptide 5 was built for 7 out of 11 residues (KENRWIL) according to the electron density. The omit map (blue mesh) of peptide 5 (in pink sticks) is contoured at 3σ. SAH is depicted in stick, the radical SAM [4Fe-4S] and AuxI clusters are shown as spheres. b, Peptide 5 fold. The peptide is shown in salmon (chain C) and orange (chain D) and colored by atom type. Intramolecular interactions are depicted in black dashed line.

Extended Data Fig. 8 Structures of C223A EpeE mutant bound with peptides 5 and 6.

a, Superimposition of EpeE WT in complex with peptide 5 (pale cyan) and EpeE C223A in complex with peptide 5 (bright orange; r.m.s.d. of 0.22 Å). b, Superimposition of EpeE WT in complex with peptide 5 (pale cyan) and EpeE C223A in complex with peptide 6 (green; r.m.s.d. of 0.23 Å). c, Close-up of EpeE C223A mutant active site. The peptide 5 (left panel) was built for 9 out of 11 residues (KSKENRWIL) according to the electron density. The peptide 6 (right panel) was built for all the 11 residues (KENRWILGSGH) according to the electron density. The omit maps (blue mesh) of peptide 5 (pink sticks) and 6 (purple sticks) are contoured at 3σ. SAH (green) is depicted in stick, the radical SAM [4Fe-4S] and AuxI clusters are shown as spheres. d, Structure of EpeE C223A mutant with peptide 6 in its active site. K171 and D143 are stacking H49 from peptide 6 (left panel) while, in the substrate-free WT EpeE structure, D143 and K171 have a distinct orientation stabilized by a salt bridge (right panel). e, The presence of H49 in the structure of EpeE C223A mutant with peptide 6 provided inter-chain interactions between the two enzyme subunits.

Extended Data Fig. 9 The C223 residue in the structures of wild-type EpeE and D210A mutant.

a, Interactions involving D210 in the structure of EpeE with peptide 5. D210 provides key electrostatic interactions to the substrate (residues N41 and R42) and is stabilized by a polar bond with the protein residue T5. The distance between C223 and D210 is 5.08 Å. b, Orientations of C223 in the structure of the D210A EpeE mutant. The omit map (blue mesh) of C223 in chain A (left panel) and chain B (right panel) is contoured at 3σ. C223 was modeled as a persulfurated cysteine residue. In chain A (left panel), C223 adopted two orientations.

Extended Data Table 1 Crystallographic data and refinement statistics
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Supplementary information

Supplementary Information

Supplementary Tables 1–10.

Reporting Summary

Source data

Source Data Fig. 4

Statistical source data for Fig. 4b.

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Kubiak, X., Polsinelli, I., Chavas, L.M.G. et al. Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme. Nat Chem Biol 20, 382–391 (2024). https://doi.org/10.1038/s41589-023-01493-1

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