Mechanistic Investigations of PoyD, a Radical S-Adenosyl-l-methionine 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 S-adenosyl-l-methionine (SAM) enzymes. Here we show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide epimerization in a C-to-N 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.

The B12-Radical SAM Enzyme PoyC Catalyzes Valine Cß-Methylation 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 S-adenosylmethionine (rSAM) enzymes, PoyB and PoyC. Here we report the in vitro activity of PoyC, the first B12-dependent rSAM enzyme catalyzing peptide Cß-methylation. We show that PoyC catalyzes the formation of S-adenosylhomocysteine and 5'-deoxyadenosine and the transfer of a methyl group to l-valine residue. In addition, we demonstrate for the first time that B12-rSAM enzymes have a tightly bound MeCbl cofactor that during catalysis transfers a methyl group originating from S-adenosyl-l-methionine. Collectively, our results shed new light on polytheonamide biosynthesis and the large and emerging family of B12-rSAM enzymes.

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols.

Friedreich's ataxia is a severe neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein that stimulates iron-sulfur (Fe-S) cluster biogenesis. In mammals, the primary steps of Fe-S cluster assembly are performed by the NFS1-ISD11-ISCU complex via the formation of a persulfide intermediate on NFS1. Here we show that frataxin modulates the reactivity of NFS1 persulfide with thiols. We use maleimide-peptide compounds along with mass spectrometry to probe cysteine-persulfide in NFS1 and ISCU. Our data reveal that in the presence of ISCU, frataxin enhances the rate of two similar reactions on NFS1 persulfide: sulfur transfer to ISCU leading to the accumulation of a persulfide on the cysteine C104 of ISCU, and sulfur transfer to small thiols such as DTT, L-cysteine and GSH leading to persulfuration of these thiols and ultimately sulfide release. These data raise important questions on the physiological mechanism of Fe-S cluster assembly and point to a unique function of frataxin as an enhancer of sulfur transfer within the NFS1-ISD11-ISCU complex.

Single asparagine to arginine mutation allows PerR to switch from PerR box to fur box.

Fur family proteins, ubiquitous in prokaryotes, play a pivotal role in microbial survival and virulence in most pathogens. Metalloregulators, such as Fur and PerR, regulate the transcription of genes connected to iron homeostasis and response to oxidative stress, respectively. In Bacillus subtilis, Fur and PerR bind with high affinity to DNA sequences differing at only two nucleotides. In addition to these differences in the PerR and Fur boxes, we identify in this study a residue located on the DNA binding motif of the Fur protein that is critical to discrimination between the two close DNA sequences. Interestingly, when this residue is introduced into PerR, it lowers the affinity of PerR for its own DNA target but confers to the protein the ability to interact strongly with the Fur DNA binding sequence. The present data show how two closely related proteins have distinct biological properties just by changing a single residue.

The rotavirus nonstructural protein NSP5 coordinates a [2Fe-2S] iron-sulfur cluster that modulates interaction to RNA.

During rotavirus infection, replication and packaging of the viral genome occur in viral factories, termed viroplasms. The viral nonstructural protein NSP5 is a major building block of viroplasms; it recruits the viral polymerase VP1, the core protein VP2, and the ATPase NSP2 inside the viroplasm to form the viral replication complex. Here we report that NSP5 is a unique viral metalloprotein that coordinates a [2Fe-2S] iron-sulfur cluster as demonstrated by the metal and labile sulfide contents, UV-visible light absorption, and electron paramagnetic resonance. Point mutations in NSP5 allowed us to identify C171 and C174, arranged in a CXC motif, as essential residues for cluster coordination. When coexpressed with NSP2, an NSP5 mutant devoid of the iron-sulfur cluster still forms viroplasm-like structures. The cluster is therefore neither involved in the interaction with NSP2 nor in the formation of viroplasm-like structures and thus presumably in viroplasm formation. Finally, we show using microscale thermophoresis that the iron-sulfur cluster modulates the affinity of NSP5 for single-stranded RNA. Because the cluster is near the binding sites of both the polymerase VP1 and the ATPase NSP2, we anticipate that this cluster is crucial for NSP5 functions, in either packaging or replication of the viral genome.