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. 2023 May 10;145(18):10167-10177.
doi: 10.1021/jacs.3c00831. Epub 2023 Apr 27.

Peptide Selenocysteine Substitutions Reveal Direct Substrate-Enzyme Interactions at Auxiliary Clusters in Radical S-Adenosyl-l-methionine Maturases

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Peptide Selenocysteine Substitutions Reveal Direct Substrate-Enzyme Interactions at Auxiliary Clusters in Radical S-Adenosyl-l-methionine Maturases

Katherine W Rush et al. J Am Chem Soc. .

Abstract

Radical S-adenosyl-l-methionine (SAM) enzymes leverage the properties of one or more iron- and sulfide-containing metallocenters to catalyze complex and radical-mediated transformations. By far the most populous superfamily of radical SAM enzymes are those that, in addition to a 4Fe-4S cluster that binds and activates the SAM cofactor, also bind one or more additional auxiliary clusters (ACs) of largely unknown catalytic significance. In this report we examine the role of ACs in two RS enzymes, PapB and Tte1186, that catalyze formation of thioether cross-links in ribosomally synthesized and post-translationally modified peptides (RiPPs). Both enzymes catalyze a sulfur-to-carbon cross-link in a reaction that entails H atom transfer from an unactivated C-H to initiate catalysis, followed by formation of a C-S bond to yield the thioether. We show that both enzymes tolerate substitution of SeCys instead of Cys at the cross-linking site, allowing the systems to be subjected to Se K-edge X-ray spectroscopy. The EXAFS data show a direct interaction with the Fe of one of the ACs in the Michaelis complex, which is replaced with a Se-C interaction under reducing conditions that lead to the product complex. Site-directed deletion of the clusters in Tte1186 provide evidence for the identity of the AC. The implications of these observations in the context of the mechanism of these thioether cross-linking enzymes are discussed.

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Conflict of interest statement

The authors declare the following competing financial interest(s): Aspects of this work have been disclosed to the University of Utah, which holds patent interests in the findings.

Figures

Figure 1
Figure 1
Generalized reaction scheme for rSAM maturases. (a) SAM undergoes reductive cleavage to form dAdo· and methionine. (b) Reductive cleavage of SAM generates dAdo·, which in turn abstracts a hydrogen atom from the substrate. The formation of the product entails the loss of a second electron and a proton, and ACs have been postulated to be involved in this process. (c) PapB produces a β-cross-link between a Cys thiol and the Cβ of Asp of the PapA substrate.
Figure 2
Figure 2
Sequences and structures of representative RiPP maturase enzymes. (a) Partial alignments of CteB (5WGG, chain A), PapB (WP_019688962), and Tte1186 (WP_011025521) sequences. The Cys residues in each cluster were identified from the published structure of CteB. The Cys residues that bind the [4Fe–4S] RS cluster are highlighted in yellow (CteB: C104, C108, and C111; PapB: C119, C123, and C126; Tte1186: C104, C108, and C111). Residues in the alignment that correlate to AC1 and AC2 in CteB are highlighted in red (C344, C362, and C413) and purple (C400, C403, C409, and C432), respectively. PapB and Tte1186 conserve the Cys residues that in CteB coordinate AC1 (PapB: C352, C370, and C421; Tte1186: C344, C362, and C413) and AC2 (PapB: C408, C411, C417, and C440; Tte1186: C400, C403, C409, and C432). The sequence alignment was generated with Clustal Omega. (b) Experimentally determined structures of CteB (blue) shown along Robetta-predicted structures for PapB (gray) and CteB (salmon). The experimental and predicted PDB files were structurally aligned and visualized with Maestro. (c) Partial TIM barrel and active site of CteB showing the RS cluster, AC1, AC2, and bound SAM cofactor. The Cys residues that coordinate to AC1 are shown in thick tube representation with the [4Fe–4S] clusters shown in CPK.
Figure 3
Figure 3
PapB cross-links C19U msPapA. (a) MS of the peptide corresponding to C19U eluting from the HPLC column (see Figure S1 for an elution profile). Mass spectra of the C19U msPapA in the presence (trace a) and absence (trace c) of PapB show multiple charge states corresponding to the peptide. In addition, an envelope that is consistent with the diselenide form of the peptide is also observed in the absence of PapB. (b) Expansion of the z = 3 charge state of the C19U msPapA reveals two nested envelopes. The major envelope corresponds to a monoisotopic mass of m/z = 860.1021, which is within 1.05 ppm of the expected (m/z = 860.1012). The minor envelope matches the masses and distributions of a diselenide C19U msPapA peptide (see Figure S2 for the comparisons and discussion of the theoretical envelope for the selenol form versus diselenide form of the peptide). (c) Mass spectrum of the C19U msPapA peptide after reaction with PapB, SAM, and DTT. A mass that is consistent with a peptide–DTT adduct appears (m/z = 910.7667). (d) Expansion of the (+) PapB z = 3 charge state reveals a 2 Da shift which is consistent with a single cross-link. The major envelope shifts upon cross-linking to m/z of 859.4313, which is within 2.32 ppm of the calculated value (m/z = 859.4293) for a single cross-link. Control experiments show that the cross-linking reaction requires PapB, SAM, and NaDT (Figure S3). (e) MS/MS analysis of C19U msPapA treated with PapB. The peptide sequence is annotated with all experimentally observed b and y fragments. No fragments are seen between U19 and D23. See Figure S4 for the full MS/MS spectrum and Table S1 for a list of all observed fragments, their expected masses, and the corresponding ppm errors.
Figure 4
Figure 4
C19U msPapA selenopeptide substrate binds PapB Fe–S cluster and undergoes selenoether formation. Se K-edge EXAFS (inset) and Fourier transforms of C19U msPapA incubated with (a) reaction buffer, (b) PapB and SAM, and (c) PapB, SAM, and NaDT. Experimental data are in black, and simulations are in red. Dashed lines on the Fourier transform plots indicate interatomic distances of 2.0 and 2.4 Å corresponding to simulated distances for Se–C and Se–Fe interactions, respectively (see Table 1). In panel a, the dashed line corresponding to R = 2.4 Å is included for easy comparison to panels b and c, but no Se–Fe interaction is present. Chemical structures below each EXAFS spectrum illustrate the interpretation of the EXAFS data. Full EXAFS simulation parameters are listed in Table S5.
Figure 5
Figure 5
Se EXAFS of wildtype and ΔRS/ΔAC2 Tte1186. Se K-edge EXAFS (inset) and Fourier transform of C31U Tte1186a incubated with (a) wild-type Tte1186 and (b) ΔRS/ΔAC2 Tte1186. Experimental data are in black, and simulations are in red. Full EXAFS simulation parameters are listed in Table S5.
Figure 6
Figure 6
Tte1186 AC1 deletion perturbs Se–Fe interaction. Comparison between the experimental Fourier transforms of the wild-type Tte1186 sample (black, dashed line) and the ΔRS/ΔAC1 Tte1186 sample (blue, solid line). Full EXAFS simulation parameters are listed in Table S5. The vertical dashed line is included for visual reference.
Figure 7
Figure 7
Mechanistic paradigms for β- or γ-cross-linking radical SAM RiPP maturases. The EXAFS data shown Figures 5 and 6 as well as Tables 1 and 2 provide direct evidence in support of this mechanism. The data clearly show that the Cys residue interacts with the Fe, likely in AC 1. Under catalytic conditions in the presence of reductant, we clearly observe that the heteroatom in the substrate is disengaged with the cluster and has acquired a new bond to carbon, consistent with thioether cross-link formation.

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