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1.
Nat Chem Biol ; 20(7): 807-809, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38609538

Subject(s)
Drug Discovery , Humans
2.
ACS Chem Biol ; 11(8): 2232-43, 2016 08 19.
Article in English | MEDLINE | ID: mdl-27248686

ABSTRACT

Plantazolicin (PZN) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that exhibits extraordinarily narrow-spectrum antibacterial activity toward the causative agent of anthrax, Bacillus anthracis. During PZN biosynthesis, a cyclodehydratase catalyzes cyclization of cysteine, serine, and threonine residues in the PZN precursor peptide (BamA) to azolines. Subsequently, a dehydrogenase oxidizes most of these azolines to thiazoles and (methyl)oxazoles. The final biosynthetic steps consist of leader peptide removal and dimethylation of the nascent N-terminus. Using a heterologously expressed and purified heterocycle synthetase, the BamA peptide was processed in vitro concordant with the pattern of post-translational modification found in the naturally occurring compound. Using a suite of BamA-derived peptides, including amino acid substitutions as well as contracted and expanded substrate variants, the substrate tolerance of the heterocycle synthetase was elucidated in vitro, and the residues crucial for leader peptide binding were identified. Despite increased promiscuity compared to what was previously observed during heterologous production in E. coli, the synthetase retained exquisite selectivity in cyclization of unnatural peptides only at positions which correspond to those cyclized in the natural product. A cleavage site was subsequently introduced to facilitate leader peptide removal, yielding mature PZN variants after enzymatic or chemical dimethylation. In addition, we report the isolation and characterization of two novel PZN-like natural products that were predicted from genome sequences but whose production had not yet been observed.


Subject(s)
Oligopeptides/biosynthesis , Oligopeptides/metabolism , Amino Acid Sequence , Escherichia coli/metabolism , In Vitro Techniques , Ligases/metabolism , Oligopeptides/chemistry , Sequence Homology, Amino Acid , Substrate Specificity
3.
ACS Chem Biol ; 10(5): 1217-26, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25668590

ABSTRACT

Streptolysin S (SLS) is a post-translationally modified peptide cytolysin that is produced by the human pathogen Streptococcus pyogenes. SLS belongs to a large family of azole-containing natural products that are biosynthesized via an evolutionarily conserved pathway. SLS is an important virulence factor during S. pyogenes infections, but despite an extensive history of study, further investigations are needed to clarify several steps of its biosynthesis. To this end, chemical inhibitors of SLS biosynthesis would be valuable tools to interrogate the various maturation steps of both SLS and biosynthetically related natural products. Such chemical inhibitors could also potentially serve as antivirulence therapeutics, which in theory may alleviate the spread of antibiotic resistance. In this work, we demonstrate that FDA-approved HIV protease inhibitors, especially nelfinavir, block a key proteolytic processing step during SLS production. This inhibition was demonstrated in live S. pyogenes cells and through in vitro protease inhibition assays. A panel of 57 nelfinavir analogs was synthesized, leading to a series of compounds with improved anti-SLS activity while illuminating structure-activity relationships. Nelfinavir was also found to inhibit the maturation of other azole-containing natural products, namely those involved in listeriolysin S, clostridiolysin S, and plantazolicin production. The use of nelfinavir analogs as inhibitors of SLS production has allowed us to begin examining the proteolysis event in SLS maturation and will aid in further investigations of the biosynthesis of SLS and related natural products.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , HIV Protease Inhibitors/pharmacology , Streptolysins/antagonists & inhibitors , Amino Acid Sequence , Aspartic Acid Proteases/antagonists & inhibitors , Bacterial Proteins/biosynthesis , Molecular Sequence Data , Protease Inhibitors/pharmacology , Proteolysis , Sequence Homology, Amino Acid , Streptococcus pyogenes/drug effects , Streptococcus pyogenes/metabolism , Streptolysins/biosynthesis
4.
J Ind Microbiol Biotechnol ; 41(2): 315-31, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24142337

ABSTRACT

Natural product discovery is currently undergoing a transformation from a phenotype-driven field to a genotype-driven one. The increasing availability of genome sequences, coupled with improved techniques for identifying biosynthetic gene clusters, has revealed that secondary metabolomes are strikingly vaster than previously thought. New approaches to correlate biosynthetic gene clusters with the compounds they produce have facilitated the production and isolation of a rapidly growing collection of what we refer to as "reverse-discovered" natural products, in analogy to reverse genetics. In this review, we present an extensive list of reverse-discovered natural products and discuss seven important lessons for natural product discovery by genome-guided methods: structure prediction, accurate annotation, continued study of model organisms, avoiding genome-size bias, genetic manipulation, heterologous expression, and potential engineering of natural product analogs.


Subject(s)
Biological Products/metabolism , Genome, Microbial , Bacteria/genetics , Bacteria/metabolism , Biological Products/chemistry , Biosynthetic Pathways/genetics , Fungi/genetics , Fungi/metabolism , Genomics/methods
5.
ACS Chem Biol ; 8(9): 1998-2008, 2013 Sep 20.
Article in English | MEDLINE | ID: mdl-23823732

ABSTRACT

Plantazolicin (PZN) is a polyheterocyclic natural product derived from a ribosomal peptide that harbors remarkable antibiotic selectivity for the causative agent of anthrax, Bacillus anthracis. To simultaneously establish the structure-activity relationship of PZN and the substrate tolerance of the biosynthetic pathway, an Escherichia coli expression strain was engineered to heterologously produce PZN analogues. Variant PZN precursor genes were produced by site-directed mutagenesis and later screened by mass spectrometry to assess post-translational modification and export by E. coli. From a screen of 72 precursor peptides, 29 PZN variants were detected. This analogue collection provided insight into the selectivity of the post-translational modifying enzymes and established the boundaries of the natural biosynthetic pathway. Unlike other studied thiazole/oxazole-modified microcins, the biosynthetic machinery appeared to be finely tuned toward the production of PZN, such that the cognate enzymes did not process even other naturally occurring sequences from similar biosynthetic clusters. The modifying enzymes were exquisitely selective, installing heterocycles only at predefined positions within the precursor peptides while leaving neighboring residues unmodified. Nearly all substitutions at positions normally harboring heterocycles prevented maturation of a PZN variant, though some exceptions were successfully produced lacking a heterocycle at the penultimate residue. No variants containing additional heterocycles were detected, although several peptide sequences yielded multiple PZN variants as a result of varying oxidation states of select residues. Eleven PZN variants were produced in sufficient quantity to facilitate purification and assessment of their antibacterial activity, providing insight into the structure-activity relationship of PZN.


Subject(s)
Anti-Bacterial Agents/metabolism , Bacillus/metabolism , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Oligopeptides/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Bacillus/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biosynthetic Pathways , Codon/genetics , Escherichia coli/genetics , Genetic Engineering , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Oligopeptides/chemistry , Oligopeptides/genetics
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