In Vitro Biosynthesis and Substrate Tolerance of the Plantazolicin Family of Natural Products
| Autor: | Douglas A. Mitchell, Patricia M. Blair, Caitlin D. Deane, Alice Lin, Jonathan I. Tietz, Brandon J. Burkhart |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Signal peptide Plantazolicin Stereochemistry Peptide In Vitro Techniques Biology 010402 general chemistry 01 natural sciences Biochemistry Article Substrate Specificity Ligases Serine 03 medical and health sciences chemistry.chemical_compound Biosynthesis Escherichia coli Amino Acid Sequence Threonine Peptide sequence chemistry.chemical_classification Sequence Homology Amino Acid General Medicine 0104 chemical sciences Amino acid 030104 developmental biology chemistry Molecular Medicine Oligopeptides |
| Zdroj: | ACS Chemical Biology. 11:2232-2243 |
| ISSN: | 1554-8937 1554-8929 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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