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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- Issue date: 2020 Oct 16
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TOWARDS CONFORMATIONAL SEQUENCING OF PROTEINS: ASSIGNMENT OF SECONDARY STRUCTURES BY ANTI-PEPTIDE ANTIBODIESBeyreuther, Konrad; Schulze-Gahmen, Ursula; Bieseler, Barbara; Prinz, Heinrich; Institute for Genetics, University of Cologne Weyertal 121, D-5000 Cologne 41, F.R. Germany (1987)We performed model studies towards assignment of &-turn and a-helices to protein primary structures with antibodies. Probing of a &-turn was attempted with anti-peptide antibodies directed against the &-turn (DPGQ) of a synthetic &-turn model-peptide (IVIVIDPGQTVTY) adopting the intended conformation &-sheet-&-turn-&-sheet. The specific anti-&-turn model-peptide antibodies have a three orders of magnitude higher affinity for the &-turn containing epitope than the control Gly-peptide (GsDPGQG,, ) of random coil structure. The antibody affinity for the &-turn region (DPGQ) increases from the primary to the hyperimmune response. Although the chosen &-turn sequence is similar to parts of the animal's own proteins, self-tolerance did not raise difficulties in generating antibodies against the R-turn model-peptide. Individual putative &-turn sequences of proteins may be probed by including their sequence between the two &-sheet cartridges of the &-turn model-peptide. Helix assignment was probed with synthetic model peptides of extended conformation including only the superimposed residues of a putative helix (every fourth residue) linked by a spacer amino acid residue (alanine throughout or the corresponding third residue of the sequence to be tested) in order to adjust the translation of the relevant residues of the model-peptide to the helical pitch. The anti-helix modelpeptide antibodies were shown by Western blotting to react in a sequence-specific manner with the corresponding model protein lactose permease of E.coli. "Conformational sequencing" i.e. sequence assignments of secondary structures by anti-peptide antibodies now seems feasable for &-turn regions and helices of proteins of known sequence.
Multiple antibody targets on herpes B glycoproteins B and D identified by screening sera of infected rhesus macaques with peptide microarrays.Hotop, Sven-Kevin; Abd El Wahed, Ahmed; Beutling, Ulrike; Jentsch, Dieter; Motzkus, Dirk; Frank, Ronald; Hunsmann, Gerhard; Stahl-Hennig, Christiane; Fritz, Hans-Joachim; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014)Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1) is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any) in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV). 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs) were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test.
The N‐terminal peptide of the transglutaminase‐activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross‐linking sitesJuettner, Norbert E.; Schmelz, Stefan; Anderl, Anita; Colin, Felix; Classen, Moritz; Pfeifer, Felicitas; Scrima, Andrea; Fuchsbauer, Hans‐Lothar; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2019-08-29)Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC220.127.116.11; griselysin (SGMPII, SgmA), EC18.104.22.168; snapalysin (ScNP), EC22.214.171.124; streptogrisin-A (SGPA), EC126.96.36.199; streptogrisin-B (SGPB), EC188.8.131.52; subtilisin BPN', EC184.108.40.206; transglutaminase, EC220.127.116.11; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC18.104.22.168. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).