Synthesis of Plantazolicin Analogues Enables Dissection of Ligand Binding Interactions of a Highly Selective Methyltransferase

A convergent strategy for the synthesis of truncated analogues of plantazolicin (PZN), a member of the thiazole/oxazole-modified microcin (TOMM) class of natural products, has been developed. These <i>N</i>-terminal mono-, tri-, and pentazole substructures of PZN were utilized to probe the substrate requirements and thermodynamic ligand binding parameters of an unusually selective PZN methyltransferase (BamL) by isothermal titration calorimetry. Our results demonstrate that the presence of a single <i>N</i>-terminal azole permits efficient processing by BamL; however, the substrate binding becomes stronger with increased polyazole chain length

Insights into Methyltransferase Specificity and Bioactivity of Derivatives of the Antibiotic Plantazolicin

Peptide antibiotics represent a class of conformationally constrained natural products of growing pharmaceutical interest. Plantazolicin (PZN) is a linear, polyheterocyclic natural product with highly selective and potent activity against the anthrax-causing bacterium, <i>Bacillus anthracis</i>. The bioactivity of PZN is contingent on dimethylation of its <i>N</i>-terminal Arg residue by an <i>S</i>-adenosylmethionine-dependent methyltransferase. Here, we explore the substrate tolerances of two homologous PZN methyltransferases by carrying out kinetic analyses of the enzymes against a synthetic panel of truncated PZN analogs containing the <i>N</i>-terminal Arg residue. X-ray cocrystal structures of the PZN methyltransferases with each of these heterocycle-containing substrates provide a rationale for understanding the strict substrate specificity of these enzymes. Kinetic studies of structure-guided, site-specific variants allowed for the assignment of residues governing catalysis and substrate scope. Microbiological testing further revealed that upon dimethylation of the <i>N</i>-terminal Arg, a pentaheterocyclized PZN analog retained potent anti-<i>B. anthracis</i> activity, nearly equal to that of full-length PZN. These studies may be useful in the biosynthetic engineering of natural product analogs with different bioactivity profiles, as demonstrated by our identification of a truncated plantazolicin derivative that is active against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA)

<i>In Vitro</i> Biosynthesis and Substrate Tolerance of the Plantazolicin Family of Natural Products

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, <i>Bacillus anthracis</i>. 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 <i>N</i>-terminus. Using a heterologously expressed and purified heterocycle synthetase, the BamA peptide was processed <i>in vitro</i> 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 <i>in vitro</i>, and the residues crucial for leader peptide binding were identified. Despite increased promiscuity compared to what was previously observed during heterologous production in <i>E. coli</i>, 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

Plantazolicin Is an Ultranarrow-Spectrum Antibiotic That Targets the Bacillus anthracis Membrane

Plantazolicin (PZN) is a ribosomally synthesized and post-translationally modified natural product from Bacillus methylotrophicus FZB42 and Bacillus pumilus. Extensive tailoring to 12 of the 14 amino acid residues in the mature natural product endows PZN with not only a rigid, polyheterocyclic structure, but also antibacterial activity. Here we report the remarkably discriminatory activity of PZN toward Bacillus anthracis, which rivals a previously described gamma (γ) phage lysis assay in distinguishing <i>B. anthracis</i> from other members of the Bacillus cereus group. We evaluate the underlying cause of this selective activity by measuring the RNA expression profile of PZN-treated <i>B. anthracis</i>, which revealed significant up-regulation of genes within the cell envelope stress response. PZN depolarizes the <i>B. anthracis</i> membrane like other cell envelope-acting compounds but uniquely localizes to distinct foci within the envelope. Selection and whole-genome sequencing of PZN-resistant mutants of <i>B. anthracis</i> implicate a relationship between the action of PZN and cardiolipin (CL) within the membrane. Exogenous CL increases the potency of PZN in wild type <i>B. anthracis</i> and promotes the incorporation of fluorescently tagged PZN in the cell envelope. We propose that PZN localizes to and exacerbates structurally compromised regions of the bacterial membrane, which ultimately results in cell lysis