Total in Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway

authored by

Asfandyar Sikandar, Maria Lopatniuk, Andriy Luzhetskyy, Rolf Müller, Jesko Koehnke

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.

External Organisation(s)

Helmholtz Centre for Infection Research (HZI)
Saarland University
University of Glasgow

Type

Article

Journal

Journal of the American Chemical Society

Volume

144

Pages

5136-5144

No. of pages

9

ISSN

0002-7863

Publication date

23.03.2022

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Catalysis, General Chemistry, Biochemistry, Colloid and Surface Chemistry

Sustainable Development Goals

SDG 3 - Good Health and Well-being

Electronic version(s)

http://hdl.handle.net/10033/623172 (Access: Open)
https://doi.org/10.1021/jacs.2c00402 (Access: Closed)