The core of the matter Koehnke, Jesko
Nature chemical biology,
11/2021, Volume:
17, Issue:
11
Journal Article
Peer reviewed
Graspetides are an important class of ribosomal natural products with potent bioactivities. New structural information provides insights into substrate recognition and catalysis, including a rare ...glimpse into the interactions between a tailoring enzyme and the core of the precursor peptide.
Ribosomal natural products contain exquisite post-translational peptide modifications that are installed by a range of pathway-specific enzymes. We present proof of principle for a Sortase A-based ...approach that enables peptide modification by enzymes from unrelated pathways. This allowed the one-pot synthesis of a new-to-nature, hybrid ribosomal natural product.
We present a Sortase A-based approach to synthesise new-to-nature hybrid RiPPs modified using RiPP enzymes from unrelated pathways.
Covering: up to 02/2019
This review covers the role of protein-protein complexes in the biosynthesis of selected ribosomally synthesized and post-translationally modified peptide (RiPP) classes. The ...genomic organization of RiPP systems usually allows the expression of each biosynthetic enzyme as an individual unit, which is in stark contrast to the giant assembly lines found in non-ribosomal peptide and polyketide synthesis systems. Evidence is mounting however that the formation of multi-enzyme complexes is critical for efficient RiPPs biosynthesis and that these complexes may be involved in substrate channeling or conformational sampling. In some pathways, polyfunctional enzymes have evolved, which can be viewed as perpetual protein complexes. We summarize what is currently known on enzyme complexes in RiPP systems for lasso peptides, cyanobactins, linear azolic peptides, thiopeptides, and lanthipeptides.
This review covers the role of protein-protein complexes in the biosynthesis of selected ribosomally synthesized and post-translationally modified peptide (RiPP) classes.
The ribosomally synthesized and post-translationally modified peptide (RiPP) bottromycin A2 possesses potent antimicrobial activity. Its biosynthesis involves the enzymatic formation of a ...macroamidine, a process previously suggested to require the concerted efforts of a YcaO enzyme (PurCD) and an amidohydrolase (PurAH) in vivo. In vitro, PurCD alone is sufficient to catalyze formation of the macroamidine, but the process is reversible. We set out to probe the role of PurAH in macroamidine formation in vitro. We demonstrate that PurAH is highly selective for macroamidine-containing precursor peptides and cleaves C-terminal of a thiazoline, thus removing the follower peptide. After follower cleavage, macroamidine formation is irreversible, indicating PurAH as the gatekeeper of bottromycin biosynthesis. The structure of PurAH suggests residues involved in catalysis, which were probed through mutagenesis.
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IJS, KILJ, NUK, PNG, UL, UM
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 complexesThoH/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-hydroxylationa 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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IJS, KILJ, NUK, PNG, UL, UM
Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways, and many of the resulting products have potential as therapeutics. The ...ATP-dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have a conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate, suggesting that recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal that the substrate leader peptide binds to and extends the β-sheet of a conserved domain of LynD, whereas catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity, and it appears to be a conserved strategy in other peptide-modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UL, UM, UPUK
Cittilins are secondary metabolites from myxobacteria comprised of three l-tyrosines and one l-isoleucine forming a bicyclic tetrapeptide scaffold with biaryl and aryl-oxygen-aryl ether bonds. Here ...we reveal that cittilins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products, for which only the crocagins have been reported from myxobacteria. A 27 amino acid precursor peptide harbors a C-terminal four amino acid core peptide, which is enzymatically modified and finally exported to yield cittilins. The small biosynthetic gene cluster responsible for cittilin biosynthesis also encodes a cytochrome P450 enzyme and a methyltransferase, whereas a gene encoding a prolyl endopeptidase for the cleavage of the precursor peptide is located outside of the cittilin biosynthetic gene cluster. We confirm the roles of the biosynthetic genes responsible for the formation of cittilins using targeted gene inactivation and heterologous expression in Streptomyces ssp. We also report first steps toward the biochemical characterization of the proposed biosynthetic pathway in vitro. An investigation of the cellular uptake properties of cittilin A connected it to a potential biological function as an inhibitor of the prokaryotic carbon storage regulator A (CsrA).
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The YcaO superfamily of proteins catalyzes the phosphorylation of peptide backbone amide bonds, which leads to the formation of azolines and azoles in ribosomally synthesized and post-translationally ...modified peptides (RiPPs). Bottromycins are RiPPs with potent antimicrobial activity, and their biosynthetic pathway contains two divergent, stand-alone YcaO enzymes, IpoC and PurCD. From an untargeted metabolomics approach, it had been suggested that PurCD acts with a partner protein to form the 12-membered macroamidine unique to bottromycins. Here we report the biochemical characterization of IpoC and PurCD. We demonstrate that IpoC installs a cysteine-derived thiazoline, whereas PurCD alone is sufficient to create the macroamidine structure. Both enzymes are catalytically promiscuous, and we generated 10 different macroamidines. Our data provide important insights into the versatility of YcaO enzymes, their ability to utilize different nucleophiles and provide a framework for the creation of novel bottromycin derivatives with enhanced bioactivity.
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IJS, KILJ, NUK, PNG, UL, UM
The natural product carolacton is a macrolide keto-carboxylic acid produced by the myxobacterium Sorangium cellulosum, and was originally described as an antibacterial compound. Here we show that ...carolacton targets FolD, a key enzyme from the folate-dependent C1 metabolism. We characterize the interaction between bacterial FolD and carolacton biophysically, structurally and biochemically. Carolacton binds FolD with nanomolar affinity, and the crystal structure of the FolD-carolacton complex reveals the mode of binding. We show that the human FolD orthologs, MTHFD1 and MTHFD2, are also inhibited in the low nM range, and that micromolar concentrations of carolacton inhibit the growth of cancer cell lines. As mitochondrial MTHFD2 is known to be upregulated in cancer cells, it may be possible to use carolacton as an inhibitor tool compound to assess MTHFD2 as an anti-cancer target.
D-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of L-amino acids into their D-counterparts is rare and chemically ...challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a D-aspartate (D-Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual α/β-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of L-Asp to D-Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH-substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites.
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