Dataset Information

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether-Bond-Forming Sactisynthase.

ABSTRACT: Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α-carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in-depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA-mediated formation of thioether bridges, which were identified mass-spectrometrically. In a proof-of-principle study, we re-engineered subtilosin A to a thioether-bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides.

SUBMITTER: Ali A

PROVIDER: S-EPMC9828075 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether-Bond-Forming Sactisynthase.

Ali Ataurehman A Happel Dominic D Habermann Jan J Schoenfeld Katrin K Macarrón Palacios Arturo A Bitsch Sebastian S Englert Simon S Schneider Hendrik H Avrutina Olga O Fabritz Sebastian S Kolmar Harald H

Angewandte Chemie (International ed. in English) 20221012 45

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α-carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in-depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible ...[more]

PMID: 36049110

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Project description:The radical non-α-carbon thioether peptides (ranthipeptides) are a newly described class of ribosomally synthesized and post-translationally modified peptide (RiPP). Ranthipeptide biosynthetic gene clusters are characterized by a Cys-rich precursor peptide and a radical S-adenosylmethionine (rSAM)-dependent enzyme that forms a thioether linkage between a Cys donor and an acceptor residue. Unlike the sulfur-to-α-carbon linked thioether peptides (sactipeptides), known ranthipeptides contain thioethers to either the β- or γ-carbon (i.e., non-α-carbon) of an acceptor residue. Recently, we reported the discovery of freyrasin, a ranthipeptide from Paenibacillus polymyxa, which contains six thioethers from Cys-X3-Asp motifs present in the precursor peptide (PapA). The linkages are exclusively to the β-carbon of Asp (S-Cβ). In this report, we performed mutational analysis of PapA and the cognate thioether-forming rSAM enzyme (PapB) to define the substrate scope. Using a mass spectrometry-based activity assay, our data show that PapB is intolerant toward Ala and Asn in the acceptor position but tolerates Glu-containing variants. NMR spectroscopic data of a Glu variant demonstrated that the thioether linkage was to the 4-position of Glu (S-Cγ). Furthermore, we demonstrate that PapB is intolerant to expansion and contraction of the thioether motifs (Cys-Xn-Asp, n = 2 or 4), although a minimal substrate featuring only one Cys-X3-Asp motif was competent for thioether formation. Akin to the sactipeptides, PapB was dependent on a RiPP recognition element (RRE) to bind the cognate precursor peptide, with deletion resulting in loss-of-function in vivo. The activity of PapB could be restored in vivo by supplying the excised RRE in trans. Finally, we reconstituted the activity of PapB in vitro, which led to modification of all six Cys residues in PapA. These studies provide insights into ranthipeptide biosynthesis and expand our understanding of rSAM enzyme chemistry in natural product biosynthesis.

Project description:Current interest in copper/dioxygen reactivity includes the influence of thioether sulfur ligation, as it concerns the formation, structures, and properties of derived copper-dioxygen complexes. Here, we report on the chemistry of {L-Cu(I)}2-(O2) species L = (DMM)ESE, (DMM)ESP, and (DMM)ESDP, which are N3S(thioether)-based ligands varied in the nature of a substituent on the S atom, along with a related N3O(ether) (EOE) ligand. Cu(I) and Cu(II) complexes have been synthesized and crystallographically characterized. Copper(I) complexes are dimeric in the solid state, [{L-Cu(I)}2](B(C6F5)4)2, however are shown by diffusion-ordered NMR spectroscopy to be mononuclear in solution. Copper(II) complexes with a general formulation [L-Cu(II)(X)](n+) {X = ClO4(-), n = 1, or X = H2O, n = 2} exhibit distorted square pyramidal coordination geometries and progressively weaker axial thioether ligation across the series. Oxygenation (-130 °C) of {((DMM)ESE)Cu(I)}(+) results in the formation of a trans-μ-1,2-peroxodicopper(II) species [{((DMM)ESE)Cu(II)}2(μ-1,2-O2(2-))](2+) (1(P)). Weakening the Cu-S bond via a change to the thioether donor found in (DMM)ESP leads to the initial formation of [{((DMM)ESP)Cu(II)}2(μ-1,2-O2(2-))](2+) (2(P)) that subsequently isomerizes to a bis-μ-oxodicopper(III) complex, [{((DMM)ESP)Cu(III)}2(μ-O(2-))2](2+) (2(O)), with 2(P) and 2(O) in equilibrium (K(eq) = [2(O)]/[2(P)] = 2.6 at -130 °C). Formulations for these Cu/O2 adducts were confirmed by resonance Raman (rR) spectroscopy. This solution mixture is sensitive to the addition of methylsulfonate, which shifts the equilibrium toward the bis-μ-oxo isomer. Further weakening of the Cu-S bond in (DMM)ESDP or substitution with an ether donor in (DMM)EOE leads to only a bis-μ-oxo species (3(O) and 4(O), respectively). Reactivity studies indicate that the bis-μ-oxodicopper(III) species (2(O), 3(O)) and not the trans-peroxo isomers (1(P) and 2(P)) are responsible for the observed ligand sulfoxidation. Our findings concerning the existence of the 2(P)/2(O) equilibrium contrast with previously established ligand-Cu(I)/O2 reactivity and possible implications are discussed.

Dataset Information

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether-Bond-Forming Sactisynthase.

Publications

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether-Bond-Forming Sactisynthase.

Similar Datasets

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