32 research outputs found
Insights into the Mechanism of Peptide Cyclodehydrations Achieved through the Chemoenzymatic Generation of Amide Derivatives
Current strategies for generating
peptides and proteins bearing
amide carbonyl derivatives rely on solid-phase peptide synthesis for
amide functionalization. Although such strategies have been successfully
implemented, technical limitations restrict both the length and sequence
of the synthetic fragments. Herein we report the repurposing of a
thiazole/oxazole-modified microcin (TOMM) cyclodehydratase to site-specifically
install amide backbone labels onto diverse peptide substrates, a method
we refer to as azoline-mediated peptide backbone labeling (AMPL).
This convenient chemoenzymatic strategy can generate both thioamides
and amides with isotopically labeled oxygen atoms. Moreover, we demonstrate
the first leader peptide-independent activity of a TOMM synthetase,
circumventing the requirement that sequences of interest be fused
to a leader peptide for modification. Through bioinformatics-guided
site-directed mutagenesis, we also convert a strictly dehydrogenase-dependent
TOMM azole synthetase into an azoline synthetase. This vastly expands
the spectrum of substrates modifiable by AMPL by allowing any <i>in vitro</i> reconstituted TOMM synthetase to be employed. To
demonstrate the utility of AMPL for mechanistic enzymology studies,
an <sup>18</sup>O-labeled substrate was generated to provide direct
evidence that cyclodehydrations in TOMMs occur through the phosphorylation
of the carbonyl oxygen preceding the cyclized residue. Furthermore,
we demonstrate that AMPL is a useful tool for establishing the location
of azolines both on <i>in vitro</i> modified peptides and
azoline-containing natural products
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
YcaO containing protein alignment
An alignment of all YcaO containing (D) proteins that were used in the analysis. This alignment was used to generate Figures S3 and S
Maximum likelihood tree with YcaO containing protiens
This is a Maximum likelihood tree with all the YcaO domain containing (D) proteins used in this study. This tree was used to create Figures S3 and S4, but colored differently depending on the TOMM created
Orchestration of Enzymatic Processing by Thiazole/Oxazole-Modified Microcin Dehydrogenases
Thiazole/oxazole-modified
microcins (TOMMs) comprise a structurally
diverse family of natural products with varied bioactivities linked
by the presence of posttranslationally installed thiazolÂ(in)Âe and
oxazolÂ(in)Âe heterocycles. The detailed investigation of the TOMM biosynthetic
enzymes from Bacillus sp. Al Hakam
(Balh) has provided significant insight into heterocycle biosynthesis.
Thiazoles and oxazoles are installed by the successive action of an
ATP-dependent cyclodehydratase (C- and D-protein) and a FMN-dependent
dehydrogenase (B-protein), which are responsible for azoline formation
and azoline oxidation, respectively. Although several studies have
focused on the mechanism of azoline formation, many details regarding
the role of the dehydrogenase (B-protein) in overall substrate processing
remain unknown. In this work, we evaluated the involvement of the
dehydrogenase in determining the order of ring formation as well as
the promiscuity of the Balh and microcin B17 cyclodehydratases to
accept a panel of noncognate dehydrogenases. In support of the observed
promiscuity, a fluorescence polarization assay was utilized to measure
binding of the dehydrogenase to the cyclodehydratase using the intrinsic
fluorescence of the FMN cofactor. Ultimately, the noncognate dehydrogenases
were shown to possess cyclodehydratase-independent activity. A previous
study identified a conserved Lys–Tyr motif to be important
for dehydrogenase activity. Using the tools developed in this study,
the Lys–Tyr motif was shown neither to alter complex formation
with the cyclodehydratase nor the reduction potential. Taken together
with the known crystal structure of a homologue, our data suggest
that the Lys–Tyr motif is of catalytic importance. Overall,
this study provides a greater level of insight into the complex orchestration
of enzymatic activity during TOMM biosynthesis
Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics
Thiopeptides
are members of the ribosomally synthesized and post-translationally
modified peptide family of natural products. Most characterized thiopeptides
display nanomolar potency toward Gram-positive bacteria by blocking
protein translation with several being produced at the industrial
scale for veterinary and livestock applications. Employing our custom
bioinformatics program, RODEO, we expand the thiopeptide family of
natural products by a factor of four. This effort revealed many new
thiopeptide biosynthetic gene clusters with products predicted to
be distinct from characterized thiopeptides and identified gene clusters
for previously characterized molecules of unknown biosynthetic origin.
To further validate our data set of predicted thiopeptide biosynthetic
gene clusters, we isolated and characterized a structurally unique
thiopeptide featuring a central piperidine and rare thioamide moiety.
Termed saalfelduracin, this thiopeptide displayed potent antibiotic
activity toward several drug-resistant Gram-positive pathogens. A
combination of whole-genome sequencing, comparative genomics, and
heterologous expression experiments confirmed that the thioamide moiety
of saalfelduracin is installed post-translationally by the joint action
of two proteins, TfuA and YcaO. These results reconcile the previously
unknown origin of the thioamide in two long-known thiopeptides, thiopeptin
and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic
space, we provide a roadmap for the discovery of additional members
of this natural product family
Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics
Thiopeptides
are members of the ribosomally synthesized and post-translationally
modified peptide family of natural products. Most characterized thiopeptides
display nanomolar potency toward Gram-positive bacteria by blocking
protein translation with several being produced at the industrial
scale for veterinary and livestock applications. Employing our custom
bioinformatics program, RODEO, we expand the thiopeptide family of
natural products by a factor of four. This effort revealed many new
thiopeptide biosynthetic gene clusters with products predicted to
be distinct from characterized thiopeptides and identified gene clusters
for previously characterized molecules of unknown biosynthetic origin.
To further validate our data set of predicted thiopeptide biosynthetic
gene clusters, we isolated and characterized a structurally unique
thiopeptide featuring a central piperidine and rare thioamide moiety.
Termed saalfelduracin, this thiopeptide displayed potent antibiotic
activity toward several drug-resistant Gram-positive pathogens. A
combination of whole-genome sequencing, comparative genomics, and
heterologous expression experiments confirmed that the thioamide moiety
of saalfelduracin is installed post-translationally by the joint action
of two proteins, TfuA and YcaO. These results reconcile the previously
unknown origin of the thioamide in two long-known thiopeptides, thiopeptin
and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic
space, we provide a roadmap for the discovery of additional members
of this natural product family
Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics
Thiopeptides
are members of the ribosomally synthesized and post-translationally
modified peptide family of natural products. Most characterized thiopeptides
display nanomolar potency toward Gram-positive bacteria by blocking
protein translation with several being produced at the industrial
scale for veterinary and livestock applications. Employing our custom
bioinformatics program, RODEO, we expand the thiopeptide family of
natural products by a factor of four. This effort revealed many new
thiopeptide biosynthetic gene clusters with products predicted to
be distinct from characterized thiopeptides and identified gene clusters
for previously characterized molecules of unknown biosynthetic origin.
To further validate our data set of predicted thiopeptide biosynthetic
gene clusters, we isolated and characterized a structurally unique
thiopeptide featuring a central piperidine and rare thioamide moiety.
Termed saalfelduracin, this thiopeptide displayed potent antibiotic
activity toward several drug-resistant Gram-positive pathogens. A
combination of whole-genome sequencing, comparative genomics, and
heterologous expression experiments confirmed that the thioamide moiety
of saalfelduracin is installed post-translationally by the joint action
of two proteins, TfuA and YcaO. These results reconcile the previously
unknown origin of the thioamide in two long-known thiopeptides, thiopeptin
and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic
space, we provide a roadmap for the discovery of additional members
of this natural product family
Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics
Thiopeptides
are members of the ribosomally synthesized and post-translationally
modified peptide family of natural products. Most characterized thiopeptides
display nanomolar potency toward Gram-positive bacteria by blocking
protein translation with several being produced at the industrial
scale for veterinary and livestock applications. Employing our custom
bioinformatics program, RODEO, we expand the thiopeptide family of
natural products by a factor of four. This effort revealed many new
thiopeptide biosynthetic gene clusters with products predicted to
be distinct from characterized thiopeptides and identified gene clusters
for previously characterized molecules of unknown biosynthetic origin.
To further validate our data set of predicted thiopeptide biosynthetic
gene clusters, we isolated and characterized a structurally unique
thiopeptide featuring a central piperidine and rare thioamide moiety.
Termed saalfelduracin, this thiopeptide displayed potent antibiotic
activity toward several drug-resistant Gram-positive pathogens. A
combination of whole-genome sequencing, comparative genomics, and
heterologous expression experiments confirmed that the thioamide moiety
of saalfelduracin is installed post-translationally by the joint action
of two proteins, TfuA and YcaO. These results reconcile the previously
unknown origin of the thioamide in two long-known thiopeptides, thiopeptin
and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic
space, we provide a roadmap for the discovery of additional members
of this natural product family
Selectivity, Directionality, and Promiscuity in Peptide Processing from a <i>Bacillus</i> sp. Al Hakam Cyclodehydratase
The <u>t</u>hiazole/<u>o</u>xazole-<u>m</u>odified <u>m</u>icrocins
(TOMMs) represent
a burgeoning class of ribosomal natural products decorated with thiazoles
and (methyl)Âoxazoles originating from cysteines, serines, and threonines.
The ribosomal nature of TOMMs allows for the generation of derivative
products from mutations in the amino acid sequence of the precursor
peptide, which ultimately manifest in differing structures and, sometimes,
biological functions. Employing a TOMM system for the purpose of creating
new structures and functions via combinatorial biosynthesis requires
processing machinery that can tolerate highly variable substrates.
In this study, TOMM enzymatic promiscuity was assessed using a currently
uncharacterized cluster in <i>Bacillus</i> sp. Al Hakam.
As determined by Fourier transform tandem mass spectrometry (FT-MS/MS),
azole rings were formed in both a regio- and chemoselective fashion.
Cognate and noncognate precursor peptides were modified in an overall
C- to N-terminal directionality, which to date is unique among characterized
ribosomal natural products. Studies focused on the inherent promiscuity
of the biosynthetic machinery elucidated a modest bias for glycine
at the preceding (−1) position and a remarkable flexibility
in the following (+1) position, even allowing for the incorporation
of charged amino acids and bisheterocyclization. Two unnatural substrates
were utilized as the conclusive test of substrate flexibility, of
which both were processed in a predictable fashion. A greater understanding
of substrate processing and enzymatic tolerance toward unnatural substrates
will prove beneficial when designing combinatorial libraries to screen
for artificial TOMMs that exhibit desired activities