SkfB Abstracts a Hydrogen Atom from Cα on SkfA To Initiate Thioether Cross-Link Formation - PubMed (original) (raw)

Nathan A Bruender et al. Biochemistry. 2016.

Abstract

Sulfur to α-carbon thioether-containing peptides (sactipeptides) are ribosomally synthesized post-translationally modified peptides with bacteriocidal activities. The thioether cross-link, which is required for biological activity, is installed by a member of the radical S-adenosyl-l-methionine (SAM) superfamily in the peptide substrate. Herein, we show that the radical SAM enzyme, SkfB, utilizes the 5'-deoxyadenosyl radical generated from the reductive cleavage of SAM to abstract a hydrogen atom from the α-carbon of the amino acid at position 12 in the substrate, SkfA, to initiate the installation of a thioether cross-link. The insights from this work can be applied to all radical SAM sactipeptide maturases.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Schematic of the sactipeptides subtilosin A, sporulation killing factor (SKF), and the two peptides, Trnα and Trnβ. The sulfur to α-carbon thioether crosslinks are highlighted in blue. The sidechains of the accepting residues remain unchanged.

Figure 2

Figure 2

Mass spectra of (A) dAdo or (B and C) SkfA isolated from reactions containing (a) wild-type, (b) M12[β-D3]A–, or (c) M12[U-D4]A–SkfA. (A) The mass spectra show the monoisotopic (m/z = 252.1079 – 252.1083) and the corresponding 15N (m/z = 253.1049 – 253.1056), 13C (m/z = 253.1112 – 253.1126), and 2H isotope peaks (m/z = 253.1138 – 253.1146) of dAdo. (B) Mass spectra of SkfA peptides zooming in on the +5 charge state (see Fig S4a–c for the full mass spectra). The peak indicated by • corresponds to SkfA with three carbamidomethylated cysteines, whereas the * corresponds to SkfA containing one thioether crosslink and two carbamidomethylated cysteines. (C) The deconvoluted mass spectra generated using the Xtract software (Thermo Fisher) from the full mass spectra shown in Fig S4a–c.

Scheme 1

Scheme 1

A proposed mechanism of thioether crosslink formation in sactipeptides. The dAdo• abstracts the Cα hydrogen atom from the acceptor residue to initiate catalysis to generate the captodatively stabilized radical intermediate.

Similar articles

Cited by

References

    1. Finking R, Marahiel MA. Ann Rev Microbiol. 2004;58:453–488. - PubMed
    1. Fischbach MA, Walsh CT. Chem Rev. 2006;106:3468–3496. - PubMed
    1. Arnison PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G, Camarero JA, Campopiano DJ, Challis GL, Clardy J, Cotter PD, Craik DJ, Dawson M, Dittmann E, Donadio S, Dorrestein PC, Entian KD, Fischbach MA, Garavelli JS, Goransson U, Gruber CW, Haft DH, Hemscheidt TK, Hertweck C, Hill C, Horswill AR, Jaspars M, Kelly WL, Klinman JP, Kuipers OP, Link AJ, Liu W, Marahiel MA, Mitchell DA, Moll GN, Moore BS, Muller R, Nair SK, Nes IF, Norris GE, Olivera BM, Onaka H, Patchett ML, Piel J, Reaney MJ, Rebuffat S, Ross RP, Sahl HG, Schmidt EW, Selsted ME, Severinov K, Shen B, Sivonen K, Smith L, Stein T, Sussmuth RD, Tagg JR, Tang GL, Truman AW, Vederas JC, Walsh CT, Walton JD, Wenzel SC, Willey JM, van der Donk WA. Nat Prod Rep. 2013;30:108–160. - PMC - PubMed
    1. Babasaki K, Takao T, Shimonishi Y, Kurahashi K. J Biochemistry. 1985;98:585–603. - PubMed
    1. Gonzalez-Pastor JE. Science. 2003;301:510–513. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources