Proreins in this family are named from the protein that catalyzes the formation of the thioether bond required for production of the sporulation killing factor (SKF) from SkfA. These proteins form the cysteine-methionine thioether bond found in the killing factor; the acceptor amino acid can be hydrophobic, aromatic or a small hydrophilic amino acid but not a larger hydrophilic amino acid, i.e. Met=Ala, Phe, Leu, Tyr>Asn, Ser>>Gln, Glu, Lys. The relative position of Cys and Met in the substrate cannot be inverted, in vitro the thioether bond cannot be made in the absence of the SkfA propeptide, suggesting this is the first reaction in SKF maturation. In vitro, in the absence of a second substrate, cleaves S-adenosyl-L-methionine into Met and 5'-dA.
Fluhe L, Burghaus O, Wieckowski BM, Giessen TW, Linne U, Marahiel MA
Two [4Fe-4S] clusters containing radical SAM enzyme SkfB catalyze thioether bond formation during the maturation of the sporulation killing factor
▸ Abstract
The sporulation killing factor (SKF) is a 26-residue ribosomally assembled and posttranslationally modified sactipeptide. It is produced by Bacillus subtilis 168 and plays a key role in its sporulation. Like all sactipeptides, SKF contains a thioether bond, which links the cysteine residue Cys4 with the α-carbon of the methionine residue Met12. In this study we demonstrate that this bond is generated by the two [4Fe-4S] clusters containing radical SAM enzyme SkfB, which is encoded in the skf operon. By mutational analysis of both cluster-binding sites, we were able to postulate a mechanism for thioether generation which is in agreement with that of AlbA. Furthermore, we were able to show that thioether bond formation is specific toward hydrophobic amino acids at the acceptor site. Additionally we demonstrate that generation of the thioether linkage is leader-peptide-dependent, suggesting that this reaction is the first step in SKF maturation.
J Am Chem Soc
2013;135(3):959-962
| PubMed ID:
23282011
González-Pastor JE, Hobbs EC, Losick R
Cannibalism by sporulating bacteria
▸ Abstract
Spore formation by the bacterium Bacillus subtilis is an elaborate developmental process that is triggered by nutrient limitation. Here we report that cells that have entered the pathway to sporulate produce and export a killing factor and a signaling protein that act cooperatively to block sister cells from sporulating and to cause them to lyse. The sporulating cells feed on the nutrients thereby released, which allows them to keep growing rather than to complete morphogenesis. We propose that sporulation is a stress-response pathway of last resort and that B. subtilis delays a commitment to spore formation by cannibalizing its siblings.
SkfB Abstracts a Hydrogen Atom from Cα on SkfA To Initiate Thioether Cross-Link Formation
▸ 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.
