5 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
Biosynthesis of F<sub>0</sub>, Precursor of the F<sub>420</sub> Cofactor, Requires a Unique Two Radical-SAM Domain Enzyme and Tyrosine as Substrate
Cofactors play key roles in metabolic pathways. Among
them F<sub>420</sub> has proved to be a very attractive target for
the selective
inhibition of archaea and actinobacteria. Its biosynthesis, in a unique
manner, involves a key enzyme, F<sub>0</sub>-synthase. This enzyme
is a large monomer in actinobacteria, while it is constituted of two
subunits in archaea and cyanobacteria. We report here the purification
of both types of F<sub>0</sub>-synthase and their <i>in vitro</i> activities. Our study allows us to establish that F<sub>0</sub>-synthase,
from both types, uses 5-amino-6-ribitylamino-2,4Ā(1<i>H</i>,3<i>H</i>)-pyrimidinedione and tyrosine as substrates
but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore,
our data support the fact that F<sub>0</sub>-synthase generates two
5ā²-deoxyadenosyl radicals for catalysis which is unprecedented
in reaction catalyzed by radical SAM 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
A Radical Transfer Pathway in Spore Photoproduct Lyase
Spore
photoproduct lyase (SPL) repairs a covalent UV-induced thymine
dimer, spore photoproduct (SP), in germinating endospores and is responsible
for the strong UV resistance of endospores. SPL is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme, which uses
a [4Fe-4S]<sup>+</sup> cluster to reduce SAM, generating a catalytic
5ā²-deoxyadenosyl radical (5ā²-dA<sup>ā¢</sup>).
This in turn abstracts a H atom from SP, generating an SP radical
that undergoes Ī² scission to form a repaired 5ā²-thymine
and a 3ā²-thymine allylic radical. Recent biochemical and structural
data suggest that a conserved cysteine donates a H atom to the thymine
radical, resulting in a putative thiyl radical. Here we present structural
and biochemical data that suggest that two conserved tyrosines are
also critical in enzyme catalysis. One [Y99<sub>(<i>Bs</i>)</sub> in <i>Bacillus subtilis</i> SPL] is downstream
of the cysteine, suggesting that SPL uses a novel hydrogen atom transfer
(HAT) pathway with a pair of cysteine and tyrosine residues to regenerate
SAM. The other tyrosine [Y97<sub>(<i>Bs</i>)</sub>] has
a structural role to facilitate SAM binding; it may also contribute
to the SAM regeneration process by interacting with the putative <sup>ā¢</sup>Y99<sub>(<i>Bs</i>)</sub> and/or 5ā²-dA<sup>ā¢</sup> intermediates to lower the energy barrier for the
second H abstraction step. Our results indicate that SPL is the first
member of the radical SAM superfamily (comprising more than 44000
members) to bear a catalytically operating HAT chain
Radical <i>S</i>āAdenosylālāMethionine Enzyme PylB: A CāCentered Radical to Convert lāLysine into (3<i>R</i>)ā3-MethylādāOrnithine
PylB is a radical S-adenosyl-l-methionine
(SAM) enzyme predicted to convert l-lysine into (3R)-3-methyl-d-ornithine, a precursor in the biosynthesis
of the 22nd proteogenic amino acid pyrrolysine. This protein highly
resembles that of the radical SAM tyrosine and tryptophan lyases,
which activate their substrate by abstracting a H atom from the amino-nitrogen
position. Here, combining in vitro assays, analytical
methods, electron paramagnetic resonance spectroscopy, and theoretical
methods, we demonstrated that instead, PylB activates its substrate
by abstracting a H atom from the CĪ³ position of l-lysine
to afford the radical-based Ī²-scission. Strikingly, we also
showed that PylB catalyzes the reverse reaction, converting (3R)-3-methyl-d-ornithine into l-lysine
and using catalytic amounts of the 5ā²-deoxyadenosyl radical.
Finally, we identified significant in vitro production
of 5ā²-thioadenosine, an unexpected shunt product that we propose
to result from the quenching of the 5ā²-deoxyadenosyl radical
species by the nearby [Fe4S4] cluster