Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition

Abstract

Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine-tryptophan cross-link, which is installed by the radical -adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon-carbon cross-link in the streptide family of natural products.

C–C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products

“…73 The Seyedsayamdost and Ando labs solved the crystal structure of SuiB (a close ortholog of StrB) from Streptococcus suis . 74 SuiB uses the precursor peptide SuiA, a homolog of StrA (Fig. 12), as a substrate.…”

“…While the SuiA core peptide is disordered in the structure, computer simulation of the core peptide structure suggested that the binding mode of the leader peptide is consistent with positioning of the core sequence in the SuiB active site. 74 Thus, binding of the leader peptide to the catalytic barrel of SuiB, rather than its RRE domain, is likely relevant to SuiB catalysis.…”

“…13. 74 In one case (mechanism A), a base residue in the active site abstracts a proton at the 7′-position. 75 In the second case (mechanism B), the amino group of the AgaA peptide substrate Lys residue serves as a general base.…”

Transcription-factor centric genome mining strategy for discovery of diverse unprecedented RiPP gene clusters

“…1) [29][30][31]. Streptide is a new family of RiPPs that are biosynthesized via a short pathway containing three genes: strA, which encodes the precursor peptide, strB, a radical S-adenosylmethionine (SAM) enzyme, and strC, an ABC transporter [32][33][34][35]. The strB enzyme is responsible for the formation of the unusual lysine-to-tryptophan crosslink.…”

RRE-Finder: A Genome-Mining Tool for Class-Independent RiPP Discovery

“…The precursor peptide binds in a cleft between the third alpha helix (α3) and the third beta strand (β3), forming an ordered, fourstranded, antiparallel beta sheet ( Figure S1). RRE domains can exist either as a discretely encoded protein (<100 residues) or as a fusion to a larger protein domain [6][7][8][9][10] . All RREs share sequence similarity to PqqD, which is a protein involved in pyrroloquinoline quinone (PQQ) cofactor synthesis-a redox cofactor produced by many prokaryotes 11 .…”