4 research outputs found
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