8 research outputs found
A General Method for Fluorescent Labeling of the N‑Termini of Lanthipeptides and Its Application to Visualize their Cellular Localization
Labeling
of natural products with biophysical probes has greatly
contributed to investigations of their modes of action and has provided
tools for visualization of their targets. A general challenge is the
availability of a suitable functional group for chemoselective modification.
We demonstrate here that an N-terminal ketone is readily introduced
into various lanthipeptides by the generation of a cryptic N-terminal
dehydro amino acid by the cognate biosynthetic enzymes. Spontaneous
hydrolysis of the N-terminal enamines results in α-ketoamides
that site-specifically react with an aminooxy-derivatized alkyne or
fluorophore. The methodology was successfully applied to prochlorosins
1.7 and 2.8, as well as the lantibiotics lacticin 481, haloduracin
α, and haloduracin β. The fluorescently modified lantibiotics
were added to bacteria, and their cellular localization was visualized
by confocal fluorescence microscopy. Lacticin 481 and haloduracin
α localized predominantly at sites of new and old cell division
as well as in punctate patterns along the long axis of rod-shaped
bacilli, similar to the localization of lipid II. On the other hand,
haloduracin β was localized nonspecifically in the absence of
haloduracin α, but formed specific patterns when coadministered
with haloduracin α. Using two-color labeling, colocalization
of both components of the two-component lantibiotic haloduracin was
demonstrated. These data with living cells supports a model in which
the α component recognizes lipid II and then recruits the β-component
Facile Removal of Leader Peptides from Lanthipeptides by Incorporation of a Hydroxy Acid
The biosynthesis of ribosomally synthesized
and post-translationally modified peptide (RiPP) natural products
typically involves a precursor peptide which contains a leader peptide
that is important for the modification process, and that is removed
in the final step by a protease. Genome mining efforts for new RiPPs
are often hampered by the lack of a general method to remove the leader
peptides. We describe here the incorporation of hydroxy acids into
the precursor peptides in <i>E. coli</i> which results in
connection of the leader peptide via an ester linkage that is readily
cleaved by simple hydrolysis. We demonstrate the method for two lantibiotics,
lacticin 481 and nukacin ISK-1
An Engineered Lantibiotic Synthetase That Does Not Require a Leader Peptide on Its Substrate
Ribosomally synthesized and post-translationally modified
peptides
are a rapidly expanding class of natural products. They are typically
biosynthesized by modification of a C-terminal segment of the precursor
peptide (the core peptide). The precursor peptide also contains an
N-terminal leader peptide that is required to guide the biosynthetic
enzymes. For bioengineering purposes, the leader peptide is beneficial
because it allows promiscuous activity of the biosynthetic enzymes
with respect to modification of the core peptide sequence. However,
the leader peptide also presents drawbacks as it needs to be present
on the core peptide and then removed in a later step. We show that
fusing the leader peptide for the lantibiotic lacticin 481 to its
biosynthetic enzyme LctM allows the protein to act on core peptides
without a leader peptide. We illustrate the use of this methodology
for preparation of improved lacticin 481 analogues containing non-proteinogenic
amino acids