3 research outputs found
The B<sub>12</sub>-Radical SAM Enzyme PoyC Catalyzes Valine C<sub>Ī²</sub>āMethylation during Polytheonamide Biosynthesis
Genomic and metagenomic
investigations have recently led to the
delineation of a novel class of natural products called ribosomally
synthesized and post-translationally modified peptides (RiPPs). RiPPs
are ubiquitous among living organisms and include pharmaceutically
relevant compounds such as antibiotics and toxins. A prominent example
is polytheonamide A, which exhibits numerous post-translational modifications,
some of which were unknown in ribosomal peptides until recently. Among
these post-translational modifications, C-methylations have been proposed
to be catalyzed by two putative radical <i>S</i>-adenosylmethionine
(rSAM) enzymes, PoyB and PoyC. Here we report the <i>in vitro</i> activity of PoyC, the first B<sub>12</sub>-dependent rSAM enzyme
catalyzing peptide C<sub>Ī²</sub>-methylation. We show that PoyC
catalyzes the formation of <i>S</i>-adenosylhomocysteine
and 5ā²-deoxyadenosine and the transfer of a methyl group to l-valine residue. In addition, we demonstrate for the first
time that B<sub>12</sub>-rSAM enzymes have a tightly bound MeCbl cofactor
that during catalysis transfers a methyl group originating from <i>S</i>-adenosyl-l-methionine. Collectively, our results
shed new light on polytheonamide biosynthesis and the large and emerging
family of B<sub>12</sub>-rSAM enzymes
Mechanistic Investigations of PoyD, a Radical <i>S</i>āAdenosylālāmethionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis
Ribosomally
synthesized and post-translationally modified peptides
(RiPPs) are a growing family of bioactive peptides. Among RiPPs, the
bacterial toxin polytheonamide A is characterized by a unique set
of post-translational modifications catalyzed by novel radical <i>S</i>-adenosyl-l-methionine (SAM) enzymes. Here we
show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide
epimerization in a <i>C</i>-to-<i>N</i> directional
manner. By combining mutagenesis experiments with labeling studies
and investigating the enzyme substrate promiscuity, we deciphered
in detail the mechanism of PoyD. We notably identified a critical
cysteine residue as a likely key H atom donor and demonstrated that
PoyD belongs to a distinct family of radical SAM peptidyl epimerases.
In addition, our study shows that the core peptide directly influences
the epimerization pattern allowing for production of peptides with
unnatural epimerization patterns
Rational Design of a Low Molecular Weight, Stable, Potent, and Long-Lasting GPR103 AzaāĪ²<sup>3</sup>āpseudopeptide Agonist
26RFa, a novel RFamide neuropeptide, is the endogenous
ligand of
the former orphan receptor GPR103. Intracerebroventricular injection
of 26RFa and its C-terminal heptapeptide, 26RFa<sub>(20ā26)</sub>, stimulates food intake in rodents. To develop potent, stable ligands
of GPR103 with low molecular weight, we have designed a series of
aza-Ī²<sup>3</sup>-containing 26RFa<sub>(20ā26)</sub> analogues
for their propensity to establish intramolecular hydrogen bonds, and
we have evaluated their ability to increase [Ca<sup>2+</sup>]<sub>i</sub> in GPR103-transfected cells. We have identified a compound,
[Cmpi<sup>21</sup>,aza-Ī²<sup>3</sup>-Hht<sup>23</sup>]Ā26RFa<sub>(21ā26)</sub>, which was 8-fold more potent than 26RFa<sub>(20ā26)</sub> in mobilizing [Ca<sup>2+</sup>]<sub>i</sub>.
This pseudopeptide was more stable in serum than 26RFa<sub>(20ā26)</sub> and exerted a longer lasting orexigenic effect in mice. This study
constitutes an important step toward the development of 26RFa analogues
that could prove useful for the treatment of feeding disorders