Total in vitro biosynthesis of the thioamitide thioholgamide and investigation of the pathway

Sikandar, A., Lopatniuk, M., Luzhetskyy, A., Müller, R. and Koehnke, J. (2022) Total in vitro biosynthesis of the thioamitide thioholgamide and investigation of the pathway. Journal of the American Chemical Society, 144(11), pp. 5136-5144. (doi: 10.1021/jacs.2c00402) (PMID:35263083)

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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.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Koehnke, Professor Jesko
Authors: Sikandar, A., Lopatniuk, M., Luzhetskyy, A., Müller, R., and Koehnke, J.
College/School:College of Science and Engineering > School of Chemistry
Journal Name:Journal of the American Chemical Society
Publisher:American Chemical Society
ISSN (Online):1520-5126
Published Online:09 March 2022
Copyright Holders:Copyright © 2022 American Chemical Society
First Published:First published in Journal of the American Chemical Society 144(11): 5136-5144
Publisher Policy:Reproduced in accordance with the publisher copyright policy

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
312847Harnessing the biosynthetic potential of bacteria to produce ribosomally synthesised natural productsJesko KoehnkeBiotechnology and Biological Sciences Research Council (BBSRC)BB/V016059/1Chemistry