Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway.
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AbstractThioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes─ThoH/I and ThoC/D. Harnessing the power of AlphaFold, we deduce that ThoD acts as a lyase and also proposes putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys, and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation─a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide.
CitationSikandar A, Lopatniuk M, Luzhetskyy A, Müller R, Koehnke J. Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway. J Am Chem Soc. 2022 Mar 23;144(11):5136-5144. doi: 10.1021/jacs.2c00402. Epub 2022 Mar 9. PMID: 35263083.
AffiliationDepartment of Microbial Natural Products (MINS), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Department of Pharmacy, Saarland University (UdS), Campus E8.1, Saarbrücken, 66123, GermanyDepartment of Pharmacy, Pharmaceutical Biotechnology, Saarland University, Saarbrücken, 66123, GermanyGerman Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Braunschweig, 38124, GermanyWorkgroup Structural Biology of Biosynthetic Enzymes, HIPS, HZI, UdS, Saarbrücken, 66123, GermanySchool of Chemistry, University of Glasgow, Glasgow, G12 8QQ, United Kingdom
PublisherACS/ American Chemical Society
DescriptionThioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes─ThoH/I and ThoC/D. Harnessing the power of AlphaFold, we deduce that ThoD acts as a lyase and also proposes putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys, and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation─a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide. © 2022 American Chemical Society. All rights reserved.
SponsorsDeutsche Forschungsgemeinschaft (DFG): MU 1254/32-1 J.K. thanks the BBSRC for support (BB/V016059/1). R.M. would like to acknowledge DFG (Leibniz Award: MU 1254/32-1).
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- Non-Heme Monooxygenase ThoJ Catalyzes Thioholgamide β-Hydroxylation.
- Authors: Sikandar A, Lopatniuk M, Luzhetskyy A, Koehnke J
- Issue date: 2020 Oct 16
- Characterization of Histidine Functionalization and Its Timing in the Biosynthesis of Ribosomally Synthesized and Posttranslationally Modified Thioamitides.
- Authors: Hu L, Qiao Y, Liu J, Zheng C, Wang X, Sun P, Gu Y, Liu W
- Issue date: 2022 Mar 16
- A ribosomally synthesised and post-translationally modified peptide containing a β-enamino acid and a macrocyclic motif.
- Authors: Wang S, Lin S, Fang Q, Gyampoh R, Lu Z, Gao Y, Clarke DJ, Wu K, Trembleau L, Yu Y, Kyeremeh K, Milne BF, Tabudravu J, Deng H
- Issue date: 2022 Aug 26
- Mechanistic Studies on Dehydration in Class V Lanthipeptides.
- Authors: Liang H, Lopez IJ, Sánchez-Hidalgo M, Genilloud O, van der Donk WA
- Issue date: 2022 Sep 16
- Structural Basis of Leader Peptide Recognition in Lasso Peptide Biosynthesis Pathway.
- Authors: Sumida T, Dubiley S, Wilcox B, Severinov K, Tagami S
- Issue date: 2019 Jul 19
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