Structure of Microcin J25, a Peptide Inhibitor of Bacterial RNA Polymerase, is a Lassoed Tail

Wilson, Kelly Anne; Kalkum, Markus; Ottesen, Jennifer J.; Yuzenkova, Yulia; Chait, Brian T.; Landick, Robert; Muir, Tom W.; Severinov, Konstantin; Darst, Seth A.

doi:10.1021/ja036756q

Cited by 224 publications

(217 citation statements)

References 31 publications

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“…The threaded tail is trapped within the cycle by the aromatic side chains of MccJ25 residues Phe 19 and Tyr 20 , which straddle the cycle (5)(6)(7)(8). The unusual lariat-protoknot structure of MccJ25 results in exceptionally high thermal stability and exceptionally high resistance to denaturation by chaotropes and organic solvents (4,8).…”

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confidence: 99%

“…The maturation of McjA is catalyzed by McjB and McjC, the products of the mcjB and mcjC genes (10). McjC is homologous to amidotransferases of the asparagine-synthetase/glutamine-hydrolase class, which catalyze transfer of ammonia or an amine from an amide donor to a carboxyl acceptor; McjC thus probably participates in formation of the lactam bond between Gly 1 and Glu 8 (5)(6)(7). McjB is a putative protease and may participate in cleavage of the McjA precursor (11).…”

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confidence: 99%

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Systematic Structure-Activity Analysis of Microcin J25

Pavlova

Mukhopadhyay

Sineva

et al. 2008

Journal of Biological Chemistry

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118

182

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Microcins are a class of small (Ͻ10 kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (1). Some microcins undergo dramatic post-translational modifications by dedicated maturation machinery (2). Post-translationally modified microcins have unusual structures and target important cellular enzymes. The object of this study, microcin J25 (MccJ25), 3 is a 21-amino acid peptide that inhibits the activity of Gram-negative bacterial RNA polymerase (RNAP) and that inhibits growth of Gram-negative bacteria (primarily or exclusively Gram-negative enterics) (3, 4). MccJ25 has an unusual lariat-protoknot ("threaded lasso") structure, consisting of an eight-amino acid cycle with backbone-side chain lactam bond between the ␣-amino group of Gly 1 and the ␥-carboxyl group of Glu 8 , followed by a 13-amino acid tail that loops back and threads through the cycle (5-7). The threaded tail is trapped within the cycle by the aromatic side chains of MccJ25 residues Phe 19 and Tyr 20 , which straddle the cycle (5-8). The unusual lariat-protoknot structure of MccJ25 results in exceptionally high thermal stability and exceptionally high resistance to denaturation by chaotropes and organic solvents (4,8).MccJ25 is produced by bacteria containing a plasmid-borne mcjABCD biosynthetic gene cluster (9). Mature MccJ25 is produced from a 58-amino acid ribosomally synthesized precursor, McjA, the product of the mcjA gene (9). Amino acid residues that become part of Mutations that confer resistance to MccJ25 in nonproducing cells map to the rpoB and rpoC genes (12, 13), which encode the RNAP ␤ and ␤Ј subunits, respectively. RNAP purified from such rpoB or rpoC mutant cells is resistant to MccJ25 in vitro, establishing that RNAP is the functional cellular target of the drug (12). Amino acid substitutions in RNAP that lead to MccJ25 resistance involve residues located in the RNAP secondary channel, which mediates entry of transcription substrates, NTPs, to the RNAP active center. It has been proposed that MccJ25 inhibits transcription, at least in part, by binding within and obstructing the RNAP secondary channel, interfering with entry of . This proposal has received support from biochemical, kinetic, and single-molecule analyses as well as from structural modeling (13,14).The fact that MccJ25 is produced from a ribosomally synthesized precursor enables one to exploit the power of genetic engineering in order to produce a library of MccJ25 mutants that can be screened for desired biological properties. Here, we report the results of such an effort. We present an essentially complete collection of point mutations in the MccJ25 coding portion of the mcjA gene, and we determine the effects of amino acid substitutions resulting from these point mutations on production of MccJ25 (comprising synthesis of MccJ25 precursor, processing of MccJ25 precursor, export of mature MccJ25, and * This work was supported, in whole or in part, by National Institutes of Health Grants GM41376 (to R. H. E.) and GM64530 (to K. S.) and by a NI...

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confidence: 99%

Systematic Structure-Activity Analysis of Microcin J25

Pavlova

Mukhopadhyay

Sineva

et al. 2008

Journal of Biological Chemistry

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118

182

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“…[20] We embarked on the cyclization (Figure 2) of linear Microcin J25, the precursor of a 21 amino acid long lasso-peptide with antibiotic properties that is naturally produced by a strain of E. coli isolated from human feces. [21] Chemical head-totail cyclization of such a long peptide is very challenging and the reaction has to be performed with extremely diluted substrate to prevent polymerization, making it difficult for scale-up. [22] Microcin J25 is hydrophobic and cosolvents such as DMSO are needed [a] Reactions were performed with 1 mM acyl donor and 3 mM acyl acceptor in 100 mL Tricine buffer (100 mM, pH 8.0).…”

Section: Resultsmentioning

confidence: 99%

Peptiligase, an Enzyme for Efficient Chemoenzymatic Peptide Synthesis and Cyclization in Water

Toplak

Nuijens²,

Quaedflieg³

et al. 2016

Adv Synth Catal

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We describe a novel, organic cosolventstable and cation-independent engineered enzyme for peptide coupling reactions. The enzyme is a variant of a stable calcium-independent mutant of subtilisin BPN', with the catalytic Ser212 mutated to Cys and Pro216 converted to Ala. The enzyme, called peptiligase, catalyzes exceptionally efficient peptide coupling in water with a surprisingly high synthesis over hydrolysis (S/H) ratio. The S/H ratio of the peptide ligation reaction is correlated to the length of the peptide substrate and proved to be > 100 for the synthesis of a 13-mer peptide, which corresponds to > 99% conversion to the ligated peptide product and < 1% hydrolytic side-reaction. Furthermore, peptiligase does not require a particular recognition motif resulting in a broadly applicable and traceless peptide ligation technology. Peptiligase is very robust, easy to produce in Bacillus subtilis, and its purification is straightforward. It shows good activity and stability in the presence of organic cosolvents and chelating or denaturing agents, enabling the ligation of poorly soluble (hydrophobic) or folded peptides. This enzyme could be useful for the (industrial) synthesis of diverse (pharmaceutical) peptides. In addition, peptiligase is able to efficiently catalyze headto-tail peptide cyclization reactions.

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“…2) is a 21-mer peptide produced by E. coli AY25 that is active against Gram-negative bacteria (125,126). It has a very unusual lassoed tail structure comprising a ring with the Cterminal end passing through the center (127)(128)(129). It inhibits abortive initiation and elongation by competitively preventing NTP uptake or binding (130).…”

Section: Microcin J25mentioning

confidence: 99%

Bacterial Transcription as a Target for Antibacterial Drug Development

Yang

Lewis

2016

Microbiol Mol Biol Rev

108

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SUMMARYTranscription, the first step of gene expression, is carried out by the enzyme RNA polymerase (RNAP) and is regulated through interaction with a series of protein transcription factors. RNAP and its associated transcription factors are highly conserved across the bacterial domain and represent excellent targets for broad-spectrum antibacterial agent discovery. Despite the numerous antibiotics on the market, there are only two series currently approved that target transcription. The determination of the three-dimensional structures of RNAP and transcription complexes at high resolution over the last 15 years has led to renewed interest in targeting this essential process for antibiotic development by utilizing rational structure-based approaches. In this review, we describe the inhibition of the bacterial transcription process with respect to structural studies of RNAP, highlight recent progress toward the discovery of novel transcription inhibitors, and suggest additional potential antibacterial targets for rational drug design.

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Structure of Microcin J25, a Peptide Inhibitor of Bacterial RNA Polymerase, is a Lassoed Tail

Cited by 224 publications

References 31 publications

Systematic Structure-Activity Analysis of Microcin J25

Systematic Structure-Activity Analysis of Microcin J25

Peptiligase, an Enzyme for Efficient Chemoenzymatic Peptide Synthesis and Cyclization in Water

Bacterial Transcription as a Target for Antibacterial Drug Development

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