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. 2024 Sep 13;10(9):3378-3391.
doi: 10.1021/acsinfecdis.4c00502. Epub 2024 Aug 27.

Discovery and Biosynthesis of Persiathiacins: Unusual Polyglycosylated Thiopeptides Active Against Multidrug Resistant Tuberculosis

Affiliations

Discovery and Biosynthesis of Persiathiacins: Unusual Polyglycosylated Thiopeptides Active Against Multidrug Resistant Tuberculosis

Yousef Dashti et al. ACS Infect Dis. .

Abstract

Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, particularly their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinomycetota extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 μM. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster (BGC). A cytochrome P450 encoded by this gene cluster catalyzes the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyze the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. One of these encodes a glycosyl transferase that was shown to catalyze attachment of a D-glucose residue to nosiheptide in vitro. The discovery of the persiathiacins and their BGC thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.

Keywords: 6-deoxysugar; RiPP; antibiotic; cytochrome P450; glycosyl transferase.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Structures of nosiheptide 1, nocathiacin I 2, and philipimycin 3, which are examples of “series e” thiopeptide antibiotics.
Figure 2
Figure 2
(A) Planar structures of persiathiacins A 4 and B 5. (B) Summary of COSY and key HMBC correlations used to assign the planar structure of persiathiacins A 4 and B 5. (C) Summary of key ROESY correlations observed for persiathiacins A 4 and B 5.
Figure 3
Figure 3
(A) Comparison of the nosiheptide (nos), nocathiacin (noc), and putative persiathiacin (per) biosynthetic gene clusters. Genes are colored as follows. Blue: thiazole formation; green: Ser/Thr dehydration; orange: DMIA formation and attachment; red: cytochromes P450; gray: 6-deoxysugar biosynthesis and attachment; brown: methyltransferases. (B) The biosynthesis of the thiopeptide core of the persiathiacins is proposed to commence with transcription and translation of perM to yield a precursor peptide comprised of an N-terminal leader peptide (LP) fused to a C-terminal core peptide (structure depicted). The core peptide undergoes a series of post-translational modifications catalyzed by several enzymes encoded by the persiathiacin biosynthetic gene cluster. See main text for further details.
Figure 4
Figure 4
The enzymes encoded by perS1perS12 are proposed to be responsible for the biosynthesis and attachment of the 6-deoxysugars to the persiathiacin aglycone. (A) Proposed pathway for assembly of TDP-6-deoxy-α-d-glucose 8, TDP-α-d-olivose 10, and TDP-α-d-amicetose 12. (B) The glycosyltransferases encoded by perS4, perS6, persS8, and perS9 are proposed to decorate the persiathiacin core peptide with l-rhamnose, 6-deoxy-d-glucose, d-olivose, and d-amicetose (persiathiacin A 4), or l-rhamnose, 6-deoxy-d-glucose, and d-olivose (persiathiacin B 5). The methyltransferases encoded by perS3, perS5, and perS7 are hypothesized to O-methylate the l-rhamnose and 6-deoxy-d-glucose residues to produce the mature antibiotics. The timing of these transformations remains to be determined.
Figure 5
Figure 5
Comparative analysis of the structures of nosiheptide 1, nocathiacin I 2, and persiathiacin A 4 and the functions of the CYPs encoded by their BGCs. Blue dashed boxes highlight hydroxyl groups proposed to be installed by homologous CYPs (NosB/NocB/PerB and NosC/NocC/PerC) encoded by all three BGCs. Red dashed circles highlight hydroxyl groups proposed to be introduced by CYPs (NocT/PerT, NocV/PerV, and NocU/PerU) encoded by the nocathiacin and persiathiacin BGCs, but not the nosiheptide BGC. A purple dashed circle highlights the hydroxyl group proposed to be introduced by the CYP encoded by perX, which is only present in the persiathiacin BGC.
Figure 6
Figure 6
(A) Reaction catalyzed by purified recombinant PerX with nosiheptide 1, in the presence of spinach ferredoxin (Fd), spinach ferredoxin reductase (Fr) and NADPH. The proposed site of oxygen atom insertion, based on the assigned function of PerX in persiathiacin biosynthesis, is highlighted in red. (B) Extracted ion chromatograms at m/z = 1222.1551 and 1238.1500, corresponding to [M + H]+ for nosiheptide and its hydroxylated derivative, respectively, from UHPLC–ESI-Q-TOF-MS analyses of: culture extracts of S. actuosus ATCC25421 expressing perX under the control of the strong constitutive ermE* promoter (top chromatogram); nosiheptide 1 after incubation for 3 h with purified recombinant PerX, Fd, Fr, and NADPH (middle chromatogram); and nosiheptide 1 after incubation for 3 h with heat-denatured PerX, Fd, Fr and NADPH (bottom chromatogram).
Figure 7
Figure 7
(A) Reaction proposed to be catalyzed by purified recombinant PerS4 with nosiheptide 1 and TDP-α-d-glucose. (B) Extracted ion chromatograms at m/z = 1384.2080, corresponding to [M + H]+ for glycosylated nosiheptide, from UHPLC–ESI-Q-TOF-MS analyses of incubations of nosiheptide 1 with TDP-α-d-glucose and PerS4 (bottom) and a control reaction containing heat-inactivated PerS4. (C) Comparison of the simulated mass spectrum for C57H53N13O17S6+ (top) with the measured spectrum of the species eluting at 16.1 min, corresponding to glycosylated nosiheptide (bottom).

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