Formation of an aminovinyl-cysteine residue in thioviridamides occurs through a path independent of known lanthionine synthetase activity

Qiu, Yanping; Liu, Jingyu; Li, Yuqing; Xue, Yanqing; Li, Wen

doi:10.1016/j.chembiol.2020.12.016

Cited by 27 publications

(67 citation statements)

References 57 publications

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“…15 Gene deletion of tvaE resulted in a significant decrease in TVA-YJ-2 production, which could largely be compensated by enhancing transcriptional level using a strong constitutive promoter. 14 In this particular case, AviMeCys formation may proceed rapidly enough to allow its formation in the absence of TvaE, albeit at much lower yields. Co-expression studies and substrate stabilization studies clearly point towards the involvement of TvaE in assisting AviMeCys formation.…”

Section: Resultsmentioning

confidence: 94%

“…Co-expression studies and substrate stabilization studies clearly point towards the involvement of TvaE in assisting AviMeCys formation. 14 The absence of reported multiple diastereoisomers of thioamitidies 7, 9, 10, 12 also suggest stereochemical control during AviMeCys formation. As both, MusF and ThoE, play a role in AviMeCys formation, it is likely that one of these proteins also controls the stereochemical outcome during cyclization.…”

Section: Resultsmentioning

confidence: 98%

“…Our results are at odds with thioviridamide and thiosparsoamide in vivo studies. 11,13,14 Although we thought it highly unlikely that the sequence of biosynthetic steps was strain specific, we decided to co-express the precursor peptide, ThoA co-express (2) and ThoF in E. coli BL21 (DE3). High-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis led to the identification of at least three modified derivatives of 2 that displayed similar mass losses reported earlier for TvaF/TvaA co-expression studies.…”

Section: Resultsmentioning

confidence: 99%

“…Enzymes responsible for installing some of the modifications have either been predicted by their homology with proteins from other RiPP pathways, or functionally characterized by co-expression studies. 11,13,14 Recently, the order of modifications has been proposed based on gene deletions and untargeted metabolomics studies (Figure S1a; we will The thioholgamide precursor peptide, composed of leader peptide (LP) and core peptide, is modified extensively. The order of PTMs is not fully understood.…”

Section: Introductionmentioning

confidence: 99%

“…11 Somewhat similarly for Tva-YJ-2, the formation of a four component complex involving the homologues of ThoC, ThoD, ThoE and ThoF has been proposed to carry out the dehydration followed by AviCys formation (Figure S2b). 14 The dehydrated and cyclized precursor peptide is then acted upon by a protease responsible for the removal of the leader peptide -it is not clear if the putative protease, ThoK, is responsible for this step. The last steps of the biosynthesis involve histidine bis-N-methylation followed by β-hydroxylation, modifications catalyzed by by ThoJ and ThoG (Figure 1a), repectively.…”

Section: Introductionmentioning

confidence: 99%

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

Sikandar

Lopatniuk

Luzhetskyy

et al. 2022

Preprint

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Thioholgamides are ribosomally synthesized and post-translationally 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 propose 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.

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Sikandar

Lopatniuk

Luzhetskyy

et al. 2022

Preprint

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Association of Radical Chemistry with LanD Flavoprotein Activity for C‐Terminal Macrocyclization of a Ribosomal Peptide by Formation of an Unsaturated Thioether Residue

et al. 2023

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LanD flavoproteins catalyze oxidative decarboxylation of the C‐terminal Cys residue of a peptide to produce an enethiol. This enethiol is highly reactive and can be coupled with an upstream dehydroamino acid through Michael addition to form S‐[2‐aminovinyl](3‐methyl)cysteine, an unsaturated thioether residue known to be characteristic of an array of C‐terminally macrocyclized, ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on a two‐stage bioinformatics mining of posttranslational modifications (PTMs) related to C‐terminal Cys processing, we report herein that LanD activity can couple with radical S‐adenosylmethionine chemistry to provide a new unsaturated thioether residue, S‐[2‐aminovinyl]‐3‐carbamoylcysteine, by conjugating the resultant enethiol with Cβ of the Asn residue in the C‐terminal NxxC motif of a peptide for macrocyclization. This study furthers our understanding of the variety of PTMs involved in creating the structure diversity of macrocyclic RiPPs.

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Association of Radical Chemistry with LanD Flavoprotein Activity for C‐Terminal Macrocyclization of a Ribosomal Peptide by Formation of an Unsaturated Thioether Residue

et al. 2023

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Formation of an aminovinyl-cysteine residue in thioviridamides occurs through a path independent of known lanthionine synthetase activity

Cited by 27 publications

References 57 publications

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

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

Association of Radical Chemistry with LanD Flavoprotein Activity for C‐Terminal Macrocyclization of a Ribosomal Peptide by Formation of an Unsaturated Thioether Residue

Association of Radical Chemistry with LanD Flavoprotein Activity for C‐Terminal Macrocyclization of a Ribosomal Peptide by Formation of an Unsaturated Thioether Residue

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