Expanding the chemical diversity of lasso peptide MccJ25 with genetically encoded noncanonical amino acids

Piscotta, Frank J.; Tharp, Jeffery M.; Liu, Wenshe Ray; Link, A. James

doi:10.1039/c4cc07778d

Cited by 67 publications

(56 citation statements)

References 46 publications

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“…We also found a small open reading frame upstream of the asparagine synthase that could provide the starting peptide molecule. Lassopeptides belong to a class of natural product known as ribosomally-synthesized and post-translationally-modified peptides (RiPPs) and have been recognized as an important class of molecules because their intrinsic protease resistance gives them great potential as scaffolds for drug design (Hegemann et al, 2015; Piscotta et al, 2015). This BGC is encoded on a fragment of the genome sequence that we predict is a plasmid; this lassopeptide BGC could therefore be passed around a population of Pseudonocardia strains in a functionally dependent manner in response to environmental circumstances.…”

Section: Resultsmentioning

confidence: 99%

Genome Analysis of Two Pseudonocardia Phylotypes Associated with Acromyrmex Leafcutter Ants Reveals Their Biosynthetic Potential

et al. 2016

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The attine ants of South and Central America are ancient farmers, having evolved a symbiosis with a fungal food crop >50 million years ago. The most evolutionarily derived attines are the Atta and Acromyrmex leafcutter ants, which harvest fresh leaves to feed their fungus. Acromyrmex and many other attines vertically transmit a mutualistic strain of Pseudonocardia and use antifungal compounds made by these bacteria to protect their fungal partner against co-evolved fungal pathogens of the genus Escovopsis. Pseudonocardia mutualists associated with the attines Apterostigma dentigerum and Trachymyrmex cornetzi make novel cyclic depsipeptide compounds called gerumycins, while a mutualist strain isolated from derived Acromyrmex octospinosus makes an unusual polyene antifungal called nystatin P1. The novelty of these antimicrobials suggests there is merit in exploring secondary metabolites of Pseudonocardia on a genome-wide scale. Here, we report a genomic analysis of the Pseudonocardia phylotypes Ps1 and Ps2 that are consistently associated with Acromyrmex ants collected in Gamboa, Panama. These were previously distinguished solely on the basis of 16S rRNA gene sequencing but genome sequencing of five Ps1 and five Ps2 strains revealed that the phylotypes are distinct species and each encodes between 11 and 15 secondary metabolite biosynthetic gene clusters (BGCs). There are signature BGCs for Ps1 and Ps2 strains and some that are conserved in both. Ps1 strains all contain BGCs encoding nystatin P1-like antifungals, while the Ps2 strains encode novel nystatin-like molecules. Strains show variations in the arrangement of these BGCs that resemble those seen in gerumycin gene clusters. Genome analyses and invasion assays support our hypothesis that vertically transmitted Ps1 and Ps2 strains have antibacterial activity that could help shape the cuticular microbiome. Thus, our work defines the Pseudonocardia species associated with Acromyrmex ants and supports the hypothesis that Pseudonocardia species could provide a valuable source of new antimicrobials.

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Section: Resultsmentioning

confidence: 99%

Genome Analysis of Two Pseudonocardia Phylotypes Associated with Acromyrmex Leafcutter Ants Reveals Their Biosynthetic Potential

et al. 2016

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“…An emerging theme in the biosynthesis of RiPPs is that the maturation enzymes tend to have broad substrate specificity (2)(3)(4)(5)(6)(7)(8). Lasso peptides are a class of RiPPs, characterized by a threaded structure resembling a slipknot (9 -11).…”

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confidence: 99%

Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase

Maksimov

Koos

Zong

et al. 2015

Journal of Biological Chemistry

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Lasso peptide isopeptidase is an enzyme that specifically hydrolyzes the isopeptide bond of lasso peptides, rendering these peptides linear. To carry out a detailed structure-activity analysis of the lasso peptide isopeptidase AtxE2 from Asticcacaulis excentricus, we solved NMR structures of its substrates astexin-2 and astexin-3. Using in vitro enzyme assays, we show that the C-terminal tail portion of these peptides is dispensable with regards to isopeptidase activity. A collection of astexin-2 and astexin-3 variants with alanine substitutions at each position within the ring and the loop was constructed, and we showed that all of these peptides except for one were cleaved by the isopeptidase. Thus, much like the lasso peptide biosynthetic enzymes, lasso peptide isopeptidase has broad substrate specificity. Quantitative analysis of the cleavage reactions indicated that alanine substitutions in loop positions of these peptides led to reduced cleavage, suggesting that the loop is serving as a recognition element for the isopeptidase.Ribosomally synthesized and post-translationally modified peptides (RiPPs) 3 (1) are a diverse set of natural products that are formed by the action of maturation enzymes on a linear peptide substrate. An emerging theme in the biosynthesis of RiPPs is that the maturation enzymes tend to have broad substrate specificity (2-8). Lasso peptides are a class of RiPPs, characterized by a threaded structure resembling a slipknot (9 -11). The internal macrocycle is realized via a single isopeptide bond installed post-translationally by two maturation enzymes (12). While there is a quickly expanding literature about the lasso peptide maturation enzymes (12-14), there is little known about the catabolism of these molecules. Recently, we reported an enzyme, lasso peptide isopeptidase, that specifically hydrolyzes the isopeptide bond of lasso peptides (Fig. 1A) (15). This enzyme, related to prolyl oligopeptidases, was found in the vicinity of a lasso peptide gene cluster in the freshwater ␣-proteobacterium Asticcacaulis excentricus. This organism has two separate lasso peptide gene clusters, each with an associated isopeptidase gene (15, 16). There have been a large number of lasso peptides discovered in proteobacteria (11,17), and an isopeptidase is commonly associated with such clusters (9).We named the two isopeptidases in A. excentricus AtxE1 and AtxE2. The gene for AtxE1 is found next to the gene cluster that encodes for the biosynthesis of astexin-1, while the AtxE2 gene is located next to the gene cluster encoding astexins-2 and -3 (Fig. 1B) (15). We have previously characterized AtxE2 in vitro (15). This enzyme cleaves astexin-2 and astexin-3, but no crossreactivity was observed between AtxE2 and astexin-1. Whereas astexin-2 and astexin-3 share relatively high sequence homology (identity at 13/24 positions), the astexin-1 sequence is more divergent (Fig. 1C). In addition, we have shown that, at least for astexin-2, the thermally unthreaded variant of the peptide is not a substrate for Atx...

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“…Nevertheless, in many RiPP pathways hypervariability has been exploited extensively to create unnatural natural product libraries. For example, not only have RiPPs been engineered with sequences not found in nature, but also highly engineered derivatives carrying non-proteinogenic amino acids can be encoded as has been successfully demonstrated in the lanthipeptides [25], cyanobactins [26] and more recently in the lasso peptides [27]. …”

Section: Introductionmentioning

confidence: 99%

Combinatorial biosynthesis of RiPPs: docking with marine life

Sardar

Schmidt

2016

Current Opinion in Chemical Biology

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Ribosomally synthesized natural products are found in all forms of life. Their biosynthesis uses simple ribosomally synthesized peptides as starting materials that are transformed into complex structures via posttranslational modifications, enriched with elaborate chemical scaffolds that make them desirable as pharmacological tools. In addition, these natural products often exhibit combinatorial biosynthesis, making them attractive targets for engineering. An increasing knowledge of their biosynthetic machinery has provided key insights into their fascinating chemistry. Marine organisms have been a rich source of this class of natural products and here we review the lessons learned from marine life that enables exploitation of their potential for combinatorial engineering, opening up new routes for peptide-based drug discovery.

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Expanding the chemical diversity of lasso peptide MccJ25 with genetically encoded noncanonical amino acids

Cited by 67 publications

References 46 publications

Genome Analysis of Two Pseudonocardia Phylotypes Associated with Acromyrmex Leafcutter Ants Reveals Their Biosynthetic Potential

Genome Analysis of Two Pseudonocardia Phylotypes Associated with Acromyrmex Leafcutter Ants Reveals Their Biosynthetic Potential

Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase

Combinatorial biosynthesis of RiPPs: docking with marine life

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