Biochemical and Spectroscopic Characterization of a Radical S-Adenosyl-L-methionine Enzyme Involved in the Formation of a Peptide Thioether Cross-Link

Abstract

Peptide-derived natural products are a class of metabolites that afford the producing organism a selective advantage over other organisms in their biological niche. While the polypeptide antibiotics produced by the nonribosomal polypeptide synthetases (NRPS) are the most widely recognized, the ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging group of natural products with diverse structures and biological functions. Both the NRPS derived peptides and the RiPPs undergo extensive post-translational modifications to produce structural diversity. Here we report the first characterization of the six cysteines in forty-five (SCIFF) [Haft, D. H. and Basu M. K. (2011) J. Bacteriol. 193, 2745-2755] peptide maturase Tte1186, which is a member of the radical S-adenosyl-l-methionine (SAM) superfamily. Tte1186 catalyzes the formation of a thioether cross-link in the peptide Tte1186a encoded by an orf located upstream of the maturase, under reducing conditions in the presence of SAM. Tte1186 contains three [4Fe-4S] clusters that are indispensable for thioether cross-link formation; however, only one cluster catalyzes the reductive cleavage of SAM. Mechanistic imperatives for the reaction catalyzed by the thioether forming radical SAM maturases will be discussed.

Figure 1

(A) The mature nisin peptide has thioether crosslinks between Cys and dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues. (B) The sactipeptide subtilosin A is shown, highlighting the three thioether crosslinks. In its fully mature form, subtilosin A is circularized. The stereochemistry at the three attachment sites is shown in red. The sactipeptides are distinct from the lanthipeptides in that the thioether crosslinks are formed to the Cα of the peptide. (C) Sequence logo for the SCIFF peptides showing the conserved C-terminal sequence. The figure was generated by aligning 100 SCIFF sequences selected from Interpro family IPR023975 using the Clustal Omega multiple sequence alignment and visualized by Weblogo.^,

Figure 2

All RS enzymes use the site differentiated [4Fe-4S] cluster to catalyze the reductive cleavage of SAM. Upon reduction from the +2 to the +1 oxidation state the [4Fe-4S] mediates an inner sphere electron transfer to SAM generating the dAdo•. This strong oxidant then abstracts a H-atom from substrate to initiate a radical-mediated transformation.

Figure 3

Reductive cleavage of SAM activity assay for wildtype and Cys→Ala variants of Tte1186. SAM standard (a) elutes at ~ 25 min and 5′-dAdo standard (b) elutes at ~ 41 min. The mass spectrum of each standard is above the corresponding peak. No 5′-dAdo was observed when SAM was incubated with wild-type Tte1186 in the absence of dithionite (c), but it was observed when SAM and wild-type Tte1186 were incubated in the presence of dithionite (d). No 5′-dAdo was detected in all variants that lacked the RC (ΔRC, e; ΔRC/ΔAC1, h; and ΔRC/ΔAC2, i). Variants lacking AC1 and/or AC2 (ΔAC1, f; ΔAC2, g; and ΔAC1/ΔAC2, j) were still able to reductively cleave SAM. The elution of SAM and 5′-dAdo was monitored at 260 nm.

Figure 4

Representative mass spectra of Tte1186a isolated after incubation with Tte1186 and dithionite in the absence (black spectrum) and presence (red spectrum) of SAM. The peaks corresponding to the charge states of Tte1186a are highlighted with the gray box with the respective charge above the corresponding peak. (A) Mass spectra of Tte1186a isolated from the reactions not quenched with iodoacetamide. The spectra on the right expands the peak for the +4 charge state to show that the Tte1186a isolated from the reaction containing SAM (red spectrum) is 2.0348 amu (4 × 0.5087) lighter than peptide isolated from the reaction lacking SAM (black spectrum). (B) Mass spectra of Tte1186a that was quenched with iodoacteamide after 6 hr incubation with Tte1186 and dithionite in either the presence or absence of SAM to alkylate all free cysteine thiolates. The spectrum on the right expands on the region corresponding to the +5 charge state isotope envelope to show that the peptide isolated from the reaction containing SAM (red spectrum) is 59.033 amu (5 × 11.8066) lighter than the peptide isolated from the reaction lacking SAM (black spectrum).

Figure 5

CID MS/MS fragmentation spectrum of the +8 charge state peak of Tte1186a. (A) CID fragmentation spectrum of unmodified Tte1186a from reactions in which SAM was omitted. The four Cys residues in the sequences shown correspond to Cys 24, Cys 28, Cys 32, and Cys 36. Cys residues that were alkylated by iodoacetamide are shown in blue. The sequence shows the conserved C-terminus and that the observed b- and y-ion fragments covered majority of the C-terminus. (B) CID fragmentation spectrum of modified Tte1186a from reactions in which SAM was present. The three Cys residues (blue) correspond to Cys 24, Cys 28, and Cys 36 that are alkylated by iodoacetamide. The sequence shows the conserved C-terminus and that the observed b- and y-ion fragments covered majority of the C-terminus. No b- or y-ions were observed for the sequence between Cys 32 (red) and Thr 37 (red) due the cyclic structure imposed by a thioether crosslink. Furthermore, all b- and y-ions that contained Cys 32 through Thr 37 lacked two hydrogens and one carbamidomethyl group.

Figure 6

Mass spectra of Tte1186a after incubation with wild-type Tte1186 and the six cluster variants. Incubation with wild-type Tte1186 and SAM in the presence (a) or absence (b) of dithionite served as the positive and negative controls. The spectra focus on the [M+4H]⁴⁺ charge state peak of the Tte1186a peptide. The mass spectra of Tte1186a isolated from reactions containing each Tte1186 cluster variant was identical to the mass spectrum of the peptide isolated from the wild-type Tte1186 negative control (ΔAC1, c; ΔAC2, d; ΔRC, e; ΔRC/ΔAC2, f; ΔRC/ΔAC1, g; ΔAC1/ΔAC2, h). Only wild-type Tte1186 was able to catalyze the formation of the crosslink in Tte1186a in the presence of SAM and dithionite, as indicated by the 0.5102 amu shift of spectrum a relative to the rest of the mass spectra.

Figure 7

Continuous wave electron paramagnetic resonance (EPR) spectroscopic analysis of the FeS clusters of wild-type Tte1186 and variants under reducing conditions in the presence (A) or absence (B) of SAM. The experimental spectra (black) represent Wild-type, a; ΔAC2, b; ΔAC1, c; ΔRC, d; ΔAC1/ΔAC2, e; ΔRC/ΔAC2, f; ΔRC/ΔAC1, g. Spectral simulations (red) were carried out using least squares fitting and a minimal set of components. In the case of ΔAC1/ΔAC2, a single component was used to simulate the RS cluster in the absence (Ae) or presence (Be) of SAM. The ΔRC/ΔAC2 and ΔRC/ΔAC1variants required two species each to simulate the spectra. Spectra were recorded at 10K, with a 9.4 GHz microwave frequency, modulation amplitude of 10 G, and 100 μW microwave power. The wild-type spectra in Aa and Ba were simulated using the g-values from the double cluster variants in the following compositions: Aa, 17% RC-SAM, 17% AC1a, 26% AC2a, and 39% AC2b; Ba, 10% RC-SAM, 50% RC+SAM, 8% AC1a, 10% AC2a, and 2% AC2b.

Figure 8

Proposed mechanisms of thioether crosslink formation in lantipeptides and sactipeptides. (A) Mechanism of thioether crosslink formation in lanthipeptides. Substrate is activated to generate a reactive intermediate (Dha or Dhb), which undergoes Michael addition to generate the initial crosslink. (B) Proposed mechanism for radical-mediated thioether cross-links in sactipeptides. The distinguishing feature of this mechanism relative to those shown in Fig. S5 is formation of a reactive intermediate, such as a ketoimine, analogous to Dha or Dhb in the lantipeptide mechanism, which would be trapped from the re- or si-face to generate mixed regioselective outcomes.