18 research outputs found
Chimeric Leader Peptides for the Generation of Non-Natural Hybrid RiPP Products
Combining
biosynthetic enzymes from multiple pathways is an attractive
approach for producing molecules with desired structural features;
however, progress has been hampered by the incompatibility of enzymes
from unrelated pathways and intolerance toward alternative substrates.
Ribosomally synthesized and posttranslationally modified peptides
(RiPPs) are a diverse natural product class that employs a biosynthetic
logic that is highly amenable to engineering new compounds. RiPP biosynthetic
proteins modify their substrates by binding to a motif typically located
in the N-terminal leader region of the precursor peptide. Here, we
exploit this feature by designing leader peptides that enable recognition
and processing by multiple enzymes from unrelated RiPP pathways. Using
this broadly applicable strategy, a thiazoline-forming cyclodehydratase
was combined with enzymes from the sactipeptide and lanthipeptide
families to create new-to-nature hybrid RiPPs. We also provide insight
into design features that enable control over the hybrid biosynthesis
to optimize enzyme compatibility and establish a general platform
for engineering additional hybrid RiPPs
Identification of an Auxiliary Leader Peptide-Binding Protein Required for Azoline Formation in Ribosomal Natural Products
Thiazole/oxazole-modified
microcins (TOMMs) are a class of post-translationally
modified peptide natural products bearing azole and azoline heterocycles.
The first step in heterocycle formation is carried out by a two component
cyclodehydratase comprised of an E1 ubiquitin-activating and a YcaO
superfamily member. Recent studies have demonstrated that the YcaO
domain is responsible for cyclodehydration, while the TOMM E1 homologue
is responsible for peptide recognition during azoline formation. Although
all characterized TOMM biosynthetic clusters contain this canonical
TOMM E1 homologue (C domain), we also identified a second, highly
divergent E1 superfamily member, annotated as an Ocin-ThiF-like protein
(F protein), associated with more than 300 TOMM biosynthetic clusters.
Here we describe the <i>in vitro</i> reconstitution of a
novel TOMM cyclodehydratase from such a cluster and demonstrate that
this auxiliary protein is required for cyclodehydration. Using a combination
of biophysical techniques, we demonstrate that the F protein, rather
than the C domain, is responsible for engaging the peptide substrate.
The C domain instead appears to serve as a scaffolding protein, bringing
the catalytic YcaO domain and the peptide binding Ocin-ThiF-like protein
into proximity. Our findings provide an updated biosynthetic framework
that provides a foundation for the characterization and reconstitution
of approximately 25% of bioinformatically identifiable TOMM synthetases
<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
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Enhancer Reprogramming Promotes Pancreatic Cancer Metastasis
Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies, owing in part to its propensity for metastasis. Here, we used an organoid culture system to investigate how transcription and the enhancer landscape become altered during discrete stages of disease progression in a PDA mouse model. This approach revealed that the metastatic transition is accompanied by massive and recurrent alterations in enhancer activity. We implicate the pioneer factor FOXA1 as a driver of enhancer activation in this system, a mechanism that renders PDA cells more invasive and less anchorage-dependent for growth in vitro, as well as more metastatic in vivo. In this context, FOXA1-dependent enhancer reprogramming activates a transcriptional program of embryonic foregut endoderm. Collectively, our study implicates enhancer reprogramming, FOXA1 upregulation, and a retrograde developmental transition in PDA metastasis
Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group
In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors