The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds

Malcolmson, Steven J.; Young, Travis S.; Ruby, J. Graham; Skewes-Cox, Peter; Walsh, Christopher T.

doi:10.1073/pnas.1307111110

Cited by 59 publications

(64 citation statements)

References 38 publications

(73 reference statements)

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“…Of the 14 thiopeptide BGCs described here, three resembled BGCs for known thiopeptides (Figure 5A). bgc56 is homologous to the berninamycin BGC from Streptomyces bernensis , and one of its two putative precursor peptides is identical to that of berninamycin (Malcolmson et al, 2013). Likewise, the genetic organization and precursor peptide sequences of bgc68 and bgc66 are similar to those of the TP-1161 ( Nocardiopsis sp.…”

Section: Resultsmentioning

confidence: 99%

A Systematic Analysis of Biosynthetic Gene Clusters in the Human Microbiome Reveals a Common Family of Antibiotics

Donia

Cimermančič

Schulze

et al. 2014

Cell

585

530

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SUMMARY In complex biological systems, small molecules often mediate microbe-microbe and microbe-host interactions. Using a systematic approach, we identified 3,118 small molecule biosynthetic gene clusters (BGCs) in genomes of human-associated bacteria and studied their representation in 752 metagenomic samples from the NIH Human Microbiome Project. Remarkably, we discovered that BGCs for a class of antibiotics in clinical trials, thiopeptides, are widely distributed in genomes and metagenomes of the human microbiota. We purified and solved the structure of a new thiopeptide antibiotic, lactocillin, from a prominent member of the vaginal microbiota. We demonstrate that lactocillin has potent antibacterial activity against a range of Gram-positive vaginal pathogens, and we show that lactocillin and other thiopeptide BGCs are expressed in vivo by analyzing human metatranscriptomic sequencing data. Our findings illustrate the widespread distribution of small-molecule-encoding BGCs in the human microbiome, and they demonstrate the bacterial production of drug-like molecules in humans.

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Section: Resultsmentioning

confidence: 99%

A Systematic Analysis of Biosynthetic Gene Clusters in the Human Microbiome Reveals a Common Family of Antibiotics

Donia

Cimermančič

Schulze

et al. 2014

Cell

585

530

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“…(iii) It opens the door to the rational design and large-scale production of new thiopeptide analogs with improved pharmacological or bioactivity properties. Attempts to heterologously express diverse thiopeptide gene clusters have been met with mixed success (7,15,(38)(39)(40). In most cases, Streptomyces species, such as Streptomyces lividans and Streptomyces coelicolor, have been employed.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis

et al. 2016

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Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically dissect this pathway, we have reconstituted it in Bacillus subtilis. Successful MP1 production required promoter engineering and the reassembly of essential biosynthetic genes in a modular plasmid. The resulting system allows for rapid pathway manipulation, including protein tagging and gene deletion. We find that 8 processing proteins are sufficient for the production of MP1 and that the tailoring enzyme TclS catalyzes a C-terminal reduction step that distinguishes MP1 from its sister compound micrococcin P2. IMPORTANCEThe emergence of antibiotic resistance is one of the most urgent human health concerns of our day. A crucial component in an integrated strategy for countering antibiotic resistance is the ability to engineer pathways for the biosynthesis of natural and derivatized antimicrobial compounds. In this study, the model organism B. subtilis was employed to reconstitute and genetically modularize a 9-gene system for the biosynthesis of micrococcin, the founding member of a growing family of thiopeptide antibiotics.

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“…5) (32). Heterocycle aromatization would undergo a protease activity-independent elimination process to produce a leader peptide carboxamide and a trisubstituted pyridine (e.g., for monocyclic members thiocillins) or hydroxypyridine (e.g., for bicyclic member nosiheptide) by an additional hydroxylation (route A) (33)(34)(35)(36)(37)(38). Alternatively, reduction(s) can proceed to generate a tetrasubstituted piperidine or dehydropiperidine or imidazopiperidine via further modifications for TSR-type bicyclic thiopeptide members that bear a QA-containing side-ring system (route B) (9).…”

Section: Discussionmentioning

confidence: 99%

An α/β-hydrolase fold protein in the biosynthesis of thiostrepton exhibits a dual activity for endopeptidyl hydrolysis and epoxide ring opening/macrocyclization

Zheng

Wang

Duan

et al. 2016

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

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Thiostrepton (TSR), an archetypal bimacrocyclic thiopeptide antibiotic that arises from complex posttranslational modifications of a genetically encoded precursor peptide, possesses a quinaldic acid (QA) moiety within the side-ring system of a thiopeptide-characteristic framework. Focusing on selective engineering of the QA moiety, i.e., by fluorination or methylation, we have recently designed and biosynthesized biologically more active TSR analogs. Using these analogs as chemical probes, we uncovered an unusual indirect mechanism of TSR-type thiopeptides, which are able to act against intracellular pathogens through host autophagy induction in addition to direct targeting of bacterial ribosome. Herein, we report the accumulation of 6′-fluoro-7′, 8′-epoxy-TSR, a key intermediate in the preparation of the analog 6′-fluoro-TSR. This unexpected finding led to unveiling of the TSR maturation process, which involves an unusual dual activity of TsrI, an α/β-hydrolase fold protein, for cascade C-N bond cleavage and formation during side-ring system construction. These two functions of TsrI rely on the same catalytic triad, Ser72-His200-Asp191, which first mediates endopeptidyl hydrolysis that occurs selectively between the residues Met-1 and Ile1 for removal of the leader peptide and then triggers epoxide ring opening for closure of the QA-containing side-ring system in a regio- and stereo-specific manner. The former reaction likely requires the formation of an acyl-Ser72 enzyme intermediate; in contrast, the latter is independent of Ser72. Consequently, C-6′ fluorination of QA lowers the reactivity of the epoxide intermediate and, thereby, allows the dissection of the TsrI-associated enzymatic process that proceeds rapidly and typically is difficult to be realized during TSR biosynthesis.

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The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds

Cited by 59 publications

References 38 publications

A Systematic Analysis of Biosynthetic Gene Clusters in the Human Microbiome Reveals a Common Family of Antibiotics

A Systematic Analysis of Biosynthetic Gene Clusters in the Human Microbiome Reveals a Common Family of Antibiotics

Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis

An α/β-hydrolase fold protein in the biosynthesis of thiostrepton exhibits a dual activity for endopeptidyl hydrolysis and epoxide ring opening/macrocyclization

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