A Radical Clock Probe Uncouples H Atom Abstraction from Thioether Cross-Link Formation by the Radical S-Adenosyl-l-methionine Enzyme SkfB

Abstract

Sporulation killing factor (SKF) is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by Bacillus. SKF contains a thioether cross-link between the α-carbon at position 40 and the thiol of Cys32, introduced by a member of the radical S-adenosyl-l-methionine (SAM) superfamily, SkfB. Radical SAM enzymes employ a 4Fe-4S cluster to bind and reductively cleave SAM to generate a 5'-deoxyadenosyl radical. SkfB utilizes this radical intermediate to abstract the α-H atom at Met40 to initiate cross-linking. In addition to the cluster that binds SAM, SkfB also has an auxiliary cluster, the function of which is not known. We demonstrate that a substrate analogue with a cyclopropylglycine (CPG) moiety replacing the wild-type Met40 side chain forgoes thioether cross-linking for an alternative radical ring opening of the CPG side chain. The ring opening reaction also takes place with a catalytically inactive SkfB variant in which the auxiliary Fe-S cluster is absent. Therefore, the CPG-containing peptide uncouples H atom abstraction from thioether bond formation, limiting the role of the auxiliary cluster to promoting thioether cross-link formation. CPG proves to be a valuable tool for uncoupling H atom abstraction from peptide modification in RiPP maturases and demonstrates potential to leverage RS enzyme reactivity to create noncanonical amino acids.

Scheme 1. (A) Thioether Formation in SkfA Initiated by H Atom Abstraction at the Cα Atom of Met40 by SkfB and (B) Radical Ring Opening Catalyzed by SkfB with CPG-Containing SkfA

Figure 1

(A) Full mass spectra of CPG-SkfA incubated in the absence (black) or presence (red) of SkfB and carbamidomethylated after incubation. (B) The +4 charge state envelopes from the mass spectra in panel A are shown. The asterisk marks the average m/z of the unreacted peptide (black). Peaks with increased intensity relative the intensity of this peak are shown with up arrows, while lower m/z peaks are shown with down arrows. The changes are shown visually in the bar graph above the red trace. (C) Deconvoluted mass spectra of the samples from panel A showing the calculated [M + H]⁺ for CPG-SkfA after iodoacetamide treatment.

Figure 2

EIC and mass spectra of CPG-containing hexapeptide trypsin digest fragments of CPG-SkfA. The top panels correspond to CPG-SkfA with no SkfB prior to trypsin treatment (black traces), while the bottom panels correspond to CPG-SkfA incubated with SkfB prior to trypsin treatment (red traces). (A) EIC for the peak at m/z 644.36–644.38 and (B) EIC for the peak at m/z 645.37–645.38. Via comparison of the black and red traces, a new peak appears upon reaction with SkfB. One is assigned as the starting material, and the new peak with a longer retention time corresponds to a new product with a ²H incorporated. Chemical structures of the proposed substrate and product are shown in panel A, with D representing ²H. (C) Mass spectrum from m/z 644 to 647 of two species found and highlighted in panel B. The new peak shows that the hexapeptide from the enzyme incubation shows an increased intensity at m/z 645.38 compared to the unreacted form.

Figure 3

MS/MS spectra of the tryptic hexapeptides of CPG-SkfA that has been incubated in ²H₂O in the absence (black) and presence (red) of SkfB. The observed y, b, and z ions are indicated in the corresponding structures. Fragments containing the CPG residue are shifted by 1 amu upon being incubated with SkfB, while fragments that do not contain the CPG residue have the same m/z values as when the enzyme is omitted (see the dotted lines connecting the black and red traces).