Modularity of RiPP Enzymes Enables Designed Synthesis of Decorated Peptides

Sardar, Debosmita; Lin, Zhenjian; Schmidt, Eric W.

doi:10.1016/j.chembiol.2015.06.014

Cited by 75 publications

(145 citation statements)

References 44 publications

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“…Many RiPPs undergo enzymatic macrocyclization, as observed in several diverse classes of compounds including bottromycins (11), cyanobactins (12), lanthipeptides (13), streptides (14), lasso peptides (15), and thiopeptides (16). Understanding the biosynthetic pathways for RiPP macrocyclization continues to be important for biotechnological application, for the identification of new gene clusters, and for the production of novel RiPP derivatives (17).…”

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confidence: 99%

Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

Chekan

Estrada

Covello

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Enzymes that can catalyze the macrocyclization of linear peptide substrates have long been sought for the production of libraries of structurally diverse scaffolds via combinatorial gene assembly as well as to afford rapid in vivo screening methods. Orbitides are plant ribosomally synthesized and posttranslationally modified peptides (RiPPs) of various sizes and topologies, several of which are shown to be biologically active. The diversity in size and sequence of orbitides suggests that the corresponding macrocyclases may be ideal catalysts for production of cyclic peptides. Here we present the biochemical characterization and crystal structures of the plant enzyme PCY1 involved in orbitide macrocyclization. These studies demonstrate how the PCY1 S9A protease fold has been adapted for transamidation, rather than hydrolysis, of acyl-enzyme intermediates to yield cyclic products. Notably, PCY1 uses an unusual strategy in which the cleaved C-terminal follower peptide from the substrate stabilizes the enzyme in a productive conformation to facilitate macrocyclization of the N-terminal fragment. The broad substrate tolerance of PCY1 can be exploited as a biotechnological tool to generate structurally diverse arrays of macrocycles, including those with nonproteinogenic elements.RiPP | biosynthesis | peptide | plant | orbitide

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confidence: 99%

Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

Chekan

Estrada

Covello

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Previously, we showed that the rates of purified enzymes alone or in combination were not affected by DMAPP concentration (11). We used a series of vectors leading to different isoprene products, demonstrating that the increased yield could be observed no matter whether DMAPP, isopentenyl pyrophosphate, or farnesyl pyrophosphate was the primary product of the pathway (Fig.…”

Section: Mechanism 4: Modulation Of Protein Activity Underlies the Cymentioning

confidence: 72%

“…A mixture of patellins with 0-2 units of isoprene was produced, and prenylation increased slowly over a series of days. Therefore, we increased production of the isoprene precursor, dimethylallylpyrophosphate (DMAPP), using the vector pMBI, which contains genes (mev) that convert mevalonic acid to DMAPP (17), which in turn is a substrate for prenyltransferase TruF1 (11). Coexpression of pTru-SD and pMBI led to a great increase in prenylation that depended on the dose of added mevalonate ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The individual enzymes are slow, with the in vitro trends paralleling the in vivo observations in this study. When all enzymes are combined in vitro, intermediates accumulate, as found in E. coli (11). The best investigation of individual DG metabolic enzymes involved a comparison of broad and narrow substrate-tolerant LanM family lanthionine synthases (2,35,36).…”

Section: Discussionmentioning

confidence: 99%

“…The product of TruA is the substrate of protease TruG, which macrocyclizes the N and C termini (10). Finally, TruF1 installs one or more isoprene units on the mature macrocycle (11).…”

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Metabolic model for diversity-generating biosynthesis

Tianero

Pierce

Raghuraman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are shortlived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.natural products | secondary metabolism | biosynthesis | RiPP | cyanobactin T here are two fundamentally different types of metabolic pathways in living systems. The first are aimed to generate one or a few discrete chemicals; these comprise the majority of pathways. The second have evolved to produce large numbers of different metabolites (1, 2). Although perhaps fewer in number, this second class, which we will call "diversity generating" (DG), may be responsible for the majority of small molecules in living systems. A key difference is that the compounds produced in the latter generally have a more limited phylogenetic distribution.The metabolic pathways first elucidated were for the synthesis of essential metabolites found in all cells, such as amino acids, purines, or pyrimidines. These conventional metabolic pathways typically comprise multiple metabolic steps, with the intermediates generated in each step converted only to the final product of the pathway. DG pathways, however, do not yield a single final product. Each enzyme in a DG pathway has relaxed substrate specificity and is able to handle a variety of compounds, carrying out the same chemical transformation on different substrates.Previously, an evolutionary framework was developed to explain why some biosynthetic pathways produce many compounds (3-5). In this study we provide the first integrated overview, to our knowledge, of the multiple metabolic steps that comprise a DG biosynthetic pathway. We have uncovered striking differences in how this pathway differs from the canonical features of conventional pathways. Our results provide an initial framework for understanding how DG pathways are designed and how key features of such pathways diverge from the textbook model.We specifically examine the tru and related pat cyanobactin pathways (1, 6). These ribosomally synthesized and posttranslationally modified (RiPP) secondary metabolite pathways were identified in cyanobacterial symbionts of coral reef animals. Their expression required transfer to a model host, Escherichia coli (Fig.

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Enzymatic Macrocyclization of 1,2,3‐Triazole Peptide Mimetics

et al. 2016

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The macrocyclization of linear peptides is very often accompanied by significant improvements in their stability and biological activity. Many strategies are available for their chemical macrocyclization, however, enzyme‐mediated methods remain of great interest in terms of synthetic utility. To date, known macrocyclization enzymes have been shown to be active on both peptide and protein substrates. Here we show that the macrocyclization enzyme of the cyanobactin family, PatGmac, is capable of macrocyclizing substrates with one, two, or three 1,4‐substituted 1,2,3‐triazole moieties. The introduction of non‐peptidic scaffolds into macrocycles is highly desirable in tuning the activity and physical properties of peptidic macrocycles. We have isolated and fully characterized nine non‐natural triazole‐containing cyclic peptides, a further ten molecules are also synthesized. PatGmac has now been shown to be an effective and versatile tool for the ring closure by peptide bond formation.

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Modularity of RiPP Enzymes Enables Designed Synthesis of Decorated Peptides

Cited by 75 publications

References 44 publications

Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

Metabolic model for diversity-generating biosynthesis

Enzymatic Macrocyclization of 1,2,3‐Triazole Peptide Mimetics

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