Abstract
Sactipeptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs). They contain a unique thioether bond, referred to as a sactionine linkage, between the sulfur atom of a cysteine residue and the α-carbon of an acceptor residue. These linkages are formed via radical chemistry and are essential for the spermicidal, antifungal, and antibacterial properties of sactipeptides. Enzymes that form these linkages, called sactisynthases, are AdoMet radical enzymes in the SPASM/Twitch subgroup whose structures are incompletely characterized. Here, we present the X-ray crystal structure to 1.29-Å resolution and Mössbauer analysis of SkfB, a sactisynthase from Bacillus subtilis involved in making sporulation killing factor (SKF). We found that SkfB is a modular enzyme with an N-terminal peptide-binding domain comprising a RiPP recognition element (RRE), a middle domain that forms a classic AdoMet radical partial (β/α)6 barrel structure and displays AdoMet bound to the [4Fe-4S] cluster, and a C-terminal region characteristic of the so-called Twitch domain housing an auxiliary iron-sulfur cluster. Notably, both crystallography and Mössbauer analyses suggest that SkfB can bind a [2Fe-2S] cluster at the auxiliary cluster site, which has been observed only once before in a SPASM/Twitch auxiliary cluster site in the structure of another AdoMet radical enzyme, the pyrroloquinoline quinone biosynthesis enzyme PqqE. Taken together, our findings indicate that SkfB from B. subtilis represents a unique enzyme containing several structural features observed in other AdoMet radical enzymes.
Highlights
Sactipeptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs)
We found that SkfB is a modular enzyme with an N-terminal peptide-binding domain comprising a RiPP recognition element (RRE), a middle domain that forms a classic AdoMet radical partial (/␣)6 barrel structure and displays AdoMet bound to the [4Fe-4S] cluster, and a C-terminal region characteristic of the so-called Twitch domain housing an auxiliary iron-sulfur cluster
The central domain of SkfB folds into a partial (/␣)6 TIM barrel with six parallel -strands comprising the inner face of the barrel and six ␣-helices flanking the outside of the barrel, providing flexibility to the rigid barrel architecture (Fig. 2, A–C)
Summary
Sactipeptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) They contain a unique thioether bond, referred to as a sactionine linkage, between the sulfur atom of a cysteine residue and the ␣-carbon of an acceptor residue. Known sactisynthases include AlbA, SkfB, and presumably CteB, which install a sactionine linkage on precursor peptides SboA, SkfA, and CteA, respectively, during the maturation of the sactipeptides subtilosin A [8], sporulation killing factor [7], and thermocellin [11], respectively (see Fig. 1) These sactisynthases are members of the S-adenosyl-L-methionine (AdoMet) radical enzyme superfamily, which utilize a canonical CX3CXC motif (where is a conserved aromatic amino acid) to coordinate a [4Fe-4S] cluster, thereby named the AdoMet radical cluster [12]. AdoMet binds to the site-differentiated iron of the AdoMet radical cluster and is reductively cleaved to produce a highly reactive 5Ј-deoxyadenosyl radical (5Ј-dAdo1⁄7) species. 5Ј-dAdo1⁄7 subsequently abstracts a hydrogen atom from the substrate, forming 5Ј-deoxyadenosine and a substrate radical, which undergoes further transformation to form the desired product
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