Structural, Biochemical, and Bioinformatic Basis for Identifying Radical SAM Cyclopropyl Synthases

Abstract

The importance of radical S-adenosyl-l-methionine (RS) enzymes in the maturation of ribosomally synthesized and post-translationally modified peptides (RiPPs) continues to expand, specifically for the RS-SPASM subfamily. We recently discovered an RS-SPASM enzyme that installs a carbon-carbon bond between the geminal methyls of valine residues, resulting in the formation of cyclopropylglycine (CPG). Here, we sought to define the family of cyclopropyl (CP) synthases because of the importance of cyclopropane scaffolds in pharmaceutical development. Using RadicalSAM.org, we bioinformatically expanded the family of CP synthases and assigned unique peptide sequences to each subclade. We identified a unique RiPP biosynthetic pathway that encodes a precursor peptide, TigB, with a repeating TIGSVS motif. Using LCMS and NMR techniques, we show that the RS enzyme associated with the pathway, TigE, catalyzes the formation of a methyl-CPG from the conserved isoleucine residing in the repeating motif of TigB. Furthermore, we obtained a crystal structure of TigE, which reveals an unusual tyrosyl ligation to the auxiliary I [4Fe-4S] cluster, provided by a glycine-tyrosine-tryptophan motif unique to all CP synthases. Further, we show that this unique tyrosyl ligation is absolutely required for TigE activity. Together, our results provide insight into how CP synthases perform this unique reaction.

Figure 1

Bioinformatic analysis of the family of cyclopropyl synthases. (A) The sequence similarity network of cluster 1-1-201:AS65 from RadicalSAM.org was sorted by the associated RiPP pathway precursor peptide repeating sequence. From this, at least six distinct RiPP families that require putative cyclopropyl synthases were discovered. (B) The TIG biosynthetic pathway is composed of at least four genes, including the precursor peptide TigB, which contains a repeating TIGSVS repeating sequence.

Figure 2

Characterization of TigE. (A) SDS-PAGE gel showing that purified TigE is nearly homogeneous. (B) The appearance of a 410 nm shoulder in the absorbance spectra of reconstituted TigE suggests that [4Fe-4S] clusters are present. (C) The X-band (9.3715 GHz) CW spectrum (black) of reduced TigE at 20 K. The simulation of the data (red dashes).

Figure 3

Analysis of the TigE reaction with TigB3R. (A) Chromatogram of the unmodified (black) and modified (red) peptide TigB3R. (B) HRMS spectra of [M + 2H]²⁺ ions and the three formed products. From bottom to top: TigB3R (predicted m/z 1627.8526, observed m/z 1627.8739), P1–loss of 2 Da (predicted m/z 1626.8448, observed m/z 1626.8674), P2 - loss of 4 Da (predicted m/z 1625.8370, observed m/z 1625.8597), P3 - loss of 6 Da (predicted m/z 1624.8292, observed m/z 1624.8515).

Figure 4

Tandem mass spectrometry was employed to identify which residues TigE modified. MS/MS analysis of the P1, P2, and P3 products localized the site of the −2, −4, and −6 Da loss to isoleucine residues highlighted in red. Fragments (b- and y-) with mass loss are indicated by * (−2 Da), ** (−4 Da), and *** (−6 Da).

Figure 5

Structural elucidation of the modified TigB3R. Stacked ¹H NMR spectra for TigB3R (black) and TigE reaction products (red) in the upfield (A) and downfield (B) regions. (C, D) ¹³C HSQC strips of overlaid spectra of the unmodified (black) and modified (red) ¹³C-TigB3R. Peak assignments for modified TigB3R are differentiated by *.

Figure 6

Structure of TigE. (A) Overall structure of TigE. The β6/α6 core of the RS domain (green) responsible for binding one [4Fe-4S] cluster that coordinates one molecule of SAM (not modeled due to poor density). The C-terminal SPASM domain (orange) contains the auxiliary [4Fe-4S] clusters, Aux I and Aux II, and comprises residues 352–462. The C-terminal tail of the enzyme (orange) rests within the binding cleft of the N-terminal RRE domain. (B) Structural comparison of TigE RRE domain (left, C-terminal tail shown in orange) to that of CteB (middle, 5WGG; bound substrate peptide in yellow) and SkfB (right, 6EFN, C-terminal tail in orange). (C) The Aux I cluster is shown to be coordinated by three cysteines within the SPASM domain. The tyrosine within the GYW CP-synthase motif coordinates the fourth iron atom of the Aux I cluster.

Figure 7

Schematic representation of the proposed mechanism for TigE.