Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

Abstract

Thiopeptides are posttranslationally processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogeneous ring, thiazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, one of the more structurally complex and well-studied thiopeptides, contains a second macrocycle bearing a quinaldic acid moiety. Antibacterial, antimalarial, and anticancer properties have been described for thiostrepton A and other thiopeptides, although the molecular details for binding the cellular target in each case are not fully elaborated. We previously demonstrated that a mutation of the TsrA core peptide, Ala4Gly, supported the successful production of the corresponding thiostrepton variant. To more thoroughly probe the thiostrepton biosynthetic machinery’s tolerance toward structural variation at the fourth position of the TsrA core peptide, we report here the saturation mutagenesis of this residue using a fosmid-dependent biosynthetic engineering method and the isolation of 16 thiostrepton analogs. Several types of side chain substitutions at the fourth position of TsrA, including those that introduce polar or branched, hydrophobic residues, are accepted, albeit with varied preferences. In contrast, proline and amino acid residues inherently charged at physiological pH are not well-tolerated at the queried site by the thiostrepton biosynthetic system. These newly generated thiostrepton analogs were assessed for their antibacterial activities and abilities to inhibit the proteolytic functions of the eukaryotic 20S proteasome. We demonstrate that the identity of the fourth amino acid residue in the thiostrepton scaffold is not critical for either ribosome or proteasome inhibition.

Thiostrepton Variants Containing a Contracted Quinaldic Acid Macrocycle Result from Mutagenesis of the Second Residue

“…The Ala4Cys substitution in TsrA similarly resulted in a mixture of minor metabolites following the substitution. 34 In that case, two of the more abundant analogues, thiostreptons Ala4Cys F1 and F2, were isolated, structurally characterized, and revealed to be lanthionine (Lan)-containing diastereomers resulting from the nonenzymatic Michael addition of the Cys4 thiol on the β-carbon of Dha17. 34 It is therefore possible that spontaneous Lan residue formation from the reaction of Cys2 with at least one dehydro residue yielded multiple products.…”

“…34 In that case, two of the more abundant analogues, thiostreptons Ala4Cys F1 and F2, were isolated, structurally characterized, and revealed to be lanthionine (Lan)-containing diastereomers resulting from the nonenzymatic Michael addition of the Cys4 thiol on the β-carbon of Dha17. 34 It is therefore possible that spontaneous Lan residue formation from the reaction of Cys2 with at least one dehydro residue yielded multiple products. Multiple stereoisomers that could ensue following this nonenzymatic event could be contributing to the complex metabolite mixture in the S. laurentii NDS1/int-A2C culture extract that may also include thiostreptons bearing a free, unmodified Cys2 thiol.…”

“…These observations suggest that, in contrast to the fate of all of the Cys residues in wild-type TsrA, Cys residues at the second and fourth positions of the TsrA core peptide are not preferred substrates for thiazol(in)e ring installation by the thiostrepton cyclodehydratase and dehydrogenase. 12, 34 Alternatively, a heterocycle could be installed among the four N-terminal residues of a thiostrepton precursor but may not be efficiently processed due to the conformational restraints imparted by this modification. Despite our best efforts, the low titers of the various TsrA Ala2Cys metabolites, coupled with chemical instability and difficulties in separation of structurally similar metabolites, hindered further characterization of the thiostrepton-like Ala2Cys analogues.…”

“…MALDI MS/MS of thiostrepton analogues typically leads to loss of the QA moiety followed by the successive fragmentation of the peptide’s N-terminal residues generating ions that are diagnostic for the identity of the second residue (Figure S1). 31, 34 Although replacement of TsrA Ala2 with the linear or aromatic side chains of Met, Phe, and Tyr did permit the successful production of mature thiopeptides, these variants were not strongly favored by the thiostrepton biosynthetic system. These three analogues yielded only 1–3 mg L −1 whereas wild-type thiostrepton A and thiostrepton Ala2Gly are produced at 115 ± 35 mg L −1 and 19 ± 3 mg L −1 , respectively, in this production platform (Table S6).…”

Thiazoles in Peptides and Peptidomimetics

“…is an inhibitor of 50S ribosome and 20S proteasome, [110] as well as a valuable tool in molecular biology for gene selection in nucleotide metabolism. Although 103 had limited solubility, thiostrepton A is marketed as a drug for treating mastitis and skin infections in domestic animals.…”