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Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway.
Sikandar, Asfandyar Lopatniuk, Maria Luzhetskyy, Andriy Müller, Rolf Koehnke, Jesko
Sikandar, Asfandyar
Lopatniuk, Maria
Luzhetskyy, Andriy
Müller, Rolf
Koehnke, Jesko
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Issue Date
2022-03-09
Submitted date
2022-01-12
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Abstract
Thioholgamides 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.
Citation
Sikandar 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.
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en
Description
Thioholgamides 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.
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1520-5126
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Deutsche 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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Attribution-NonCommercial-ShareAlike 4.0 International