5 research outputs found
Biosynthetic Versatility and Coordinated Action of 5ā²-Deoxyadenosyl Radicals in Deazaflavin Biosynthesis
Coenzyme F<sub>420</sub> is a redox cofactor found in methanogens
and in various actinobacteria. Despite the major biological importance
of this cofactor, the biosynthesis of its deazaflavin core (8-hydroxy-5-deazaflavin,
F<sub>o</sub>) is still poorly understood. F<sub>o</sub> synthase,
the enzyme involved, is an unusual multidomain radical SAM enzyme
that uses two separate 5ā²-deoxyadenosyl radicals to catalyze
F<sub>o</sub> formation. In this paper, we report a detailed mechanistic
study on this complex enzyme that led us to identify (1) the hydrogen
atoms abstracted from the substrate by the two radical SAM domains,
(2) the second tyrosine-derived product, (3) the reaction product
of the CofH-catalyzed reaction, (4) the demonstration that this product
is a substrate for CofG, and (5) a stereochemical study that is consistent
with the formation of a <i>p</i>-hydroxybenzyl radical at
the CofH active site. These results enable us to propose a mechanism
for F<sub>o</sub> synthase and uncover a new catalytic motif in radical
SAM enzymology involving the use of two 5ā²-deoxyadenosyl radicals
to mediate the formation of a complex heterocycle
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
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