Construction of a Single Polypeptide that Matures and Exports the Lasso Peptide Microcin J25

Pan, Si Jia; Rajniak, Jakub; Cheung-Lee, Wai Ling; Link, A. James

doi:10.1002/cbic.201100596

Cited by 34 publications

(38 citation statements)

References 28 publications

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“…Four conserved motifs were identified in the mcjB/capB homologs ( Fig. 2), including the cysteine-histidine-aspartate catalytic triad of cysteine proteases, which is required for the function of McjB (22,23). Three motifs were found in mcjC/capC homologs ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The precursor protein McjA contains a 37-aa N-terminal leader sequence that is removed during the processing of the lasso peptide. The requisite peptidase activity for this transformation has been mapped onto the mcjB gene product (22,23). The McjC enzyme has homology to ATP-dependent amide bond-forming enzymes (10,22,23), and is proposed to catalyze the formation of the isopeptide bond in MccJ25.…”

mentioning

confidence: 99%

“…The requisite peptidase activity for this transformation has been mapped onto the mcjB gene product (22,23). The McjC enzyme has homology to ATP-dependent amide bond-forming enzymes (10,22,23), and is proposed to catalyze the formation of the isopeptide bond in MccJ25. In vitro studies on these enzymes revealed that the McjB and McjC enzymes must function in a concerted fashion to convert McjA into mature MccJ25 (21,23).…”

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confidence: 99%

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Precursor-centric genome-mining approach for lasso peptide discovery

Maksimov¹,

Pelczer²,

Link

2012

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

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136

232

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Lasso peptides are a class of ribosomally synthesized posttranslationally modified natural products found in bacteria. Currently known lasso peptides have a diverse set of pharmacologically relevant activities, including inhibition of bacterial growth, receptor antagonism, and enzyme inhibition. The biosynthesis of lasso peptides is specified by a cluster of three genes encoding a precursor protein and two enzymes. Here we develop a unique genome-mining algorithm to identify lasso peptide gene clusters in prokaryotes. Our approach involves pattern matching to a small number of conserved amino acids in precursor proteins, and thus allows for a more global survey of lasso peptide gene clusters than does homology-based genome mining. Of more than 3,000 currently sequenced prokaryotic genomes, we found 76 organisms that are putative lasso peptide producers. These organisms span nine bacterial phyla and an archaeal phylum. To provide validation of the genome-mining method, we focused on a single lasso peptide predicted to be produced by the freshwater bacterium Asticcacaulis excentricus. Heterologous expression of an engineered, minimal gene cluster in Escherichia coli led to the production of a unique lasso peptide, astexin-1. At 23 aa, astexin-1 is the largest lasso peptide isolated to date. It is also highly polar, in contrast to many lasso peptides that are primarily hydrophobic. Astexin-1 has modest antimicrobial activity against its phylogenetic relative Caulobacter crescentus. The solution structure of astexin-1 was determined revealing a unique topology that is stabilized by hydrogen bonding between segments of the peptide.bioinformatics | protein NMR

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Section: Resultsmentioning

confidence: 99%

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Precursor-centric genome-mining approach for lasso peptide discovery

Maksimov¹,

Pelczer²,

Link

2012

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

Self Cite

136

232

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“…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)(13)(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.…”

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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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“…They are synthesised by relatively small biosynthetic gene clusters which contain genes encoding a precursor peptide and enzymes that process the precursor to yield the mature form of the lasso peptide (Maksimov et al 2012;Pan et al 2012).…”

Section: Three Lasso Peptide Biosynthesis Gene Clustersmentioning

confidence: 99%

The ‘gifted’ actinomycete Streptomyces leeuwenhoekii

Castro

Razmilic

Gomez-Escribano

et al. 2018

Antonie van Leeuwenhoek

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Streptomyces leeuwenhoekii strains C34T , C38, C58 and C79 were isolated from a soil sample collected from the Chaxa Lagoon, located in the Salar de Atacama in northern Chile. These streptomycetes produce a variety of new specialised metabolites with antibiotic, anti-cancer and anti-inflammatory activities. Moreover, genome mining performed on two of these strains has revealed the presence of biosynthetic gene clusters with the potential to produce new specialised metabolites. This review focusses on this new clade of Streptomyces strains, summarises the literature and presents new information on strain C34T .

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Construction of a Single Polypeptide that Matures and Exports the Lasso Peptide Microcin J25

Cited by 34 publications

References 28 publications

Precursor-centric genome-mining approach for lasso peptide discovery

Precursor-centric genome-mining approach for lasso peptide discovery

Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase

The ‘gifted’ actinomycete Streptomyces leeuwenhoekii

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