Radical S-Adenosylmethionine Enzymes in Human Health and Disease.

Radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Of approximately 114,000 of these enzymes, 8 are known to be present in humans: MOCS1, molybdenum cofactor biosynthesis; LIAS, lipoic acid biosynthesis; CDK5RAP1, 2-methylthio-N(6)-isopentenyladenosine biosynthesis; CDKAL1, methylthio-N(6)-threonylcarbamoyladenosine biosynthesis; TYW1, wybutosine biosynthesis; ELP3, 5-methoxycarbonylmethyl uridine; and RSAD1 and viperin, both of unknown function. Aberrations in the genes encoding these proteins result in a variety of diseases. In this review, we summarize the biochemical characterization of these 8 radical S-adenosylmethionine enzymes and, in the context of human health, describe the deleterious effects that result from such genetic mutations.

Transformations of the FeS Clusters of the Methylthiotransferases MiaB and RimO, Detected by Direct Electrochemistry.

The methylthiotransferases (MTTases) represent a subfamily of the S-adenosylmethionine (AdoMet) radical superfamily of enzymes that catalyze the attachment of a methylthioether (-SCH3) moiety on unactivated carbon centers. These enzymes contain two [4Fe-4S] clusters, one of which participates in the reductive fragmentation of AdoMet to generate a 5'-deoxyadenosyl 5'-radical and the other of which, termed the auxiliary cluster, is believed to play a central role in constructing the methylthio group and attaching it to the substrate. Because the redox properties of the bound cofactors within the AdoMet radical superfamily are so poorly understood, we have examined two MTTases in parallel, MiaB and RimO, using protein electrochemistry. We resolve the redox potentials of each [4Fe-4S] cluster, show that the auxiliary cluster has a potential higher than that of the AdoMet-binding cluster, and demonstrate that upon incubation of either enzyme with AdoMet, a unique low-potential state of the enzyme emerges. Our results are consistent with a mechanism whereby the auxiliary cluster is transiently methylated during substrate methylthiolation.

Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase.

RimO is a member of the growing radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a reduced [4Fe-4S] cluster to effect reductive cleavage of the 5' C-S bond of SAM to form a 5'-deoxyadenosyl 5'-radical (5'-dA(•)) intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (-SCH3) to C3 of aspartate 89 of protein S12, one of 21 proteins that compose the 30S subunit of the bacterial ribosome. However, the exact mechanism by which this transformation takes place has remained elusive. Herein, we describe the stereochemical course of the RimO reaction. Using peptide mimics of the S12 protein bearing deuterium at the 3 pro-R or 3 pro-S positions of the target aspartyl residue, we show that RimO from Bacteroides thetaiotaomicron (Bt) catalyzes abstraction of the pro-S hydrogen atom, as evidenced by the transfer of deuterium into 5'-deoxyadenosine (5'-dAH). The observed kinetic isotope effect on H atom versus D atom abstraction is ∼1.9, suggesting that this step is at least partially rate determining. We also demonstrate that Bt RimO can utilize the flavodoxin/flavodoxin oxidoreductase/NADPH reducing system from Escherichia coli as a source of requisite electrons. Use of this in vivo reducing system decreases, but does not eliminate, formation of 5'-dAH in excess of methylthiolated product.

Trifluoroselenomethionine: A New Unnatural Amino Acid.

Trifluoroselenomethionine (TFSeM), a new unnatural amino acid, was synthesized in seven steps from N-(tert-butoxycarbonyl)-l-aspartic acid tert-butyl ester. TFSeM shows enhanced methioninase-induced cytotoxicity, relative to selenomethionine (SeM), toward HCT-116 cells derived from human colon cancer. Mechanistic explanations for this enhanced activity are computationally and experimentally examined. Comparison of TFSeM and SeM by selenium EXAFS and DFT calculations showed them to be spectroscopically and structurally very similar. Nonetheless, when two different variants of the protein GB1 were expressed in an Escherichia coli methionine auxotroph cell line in the presence of TFSeM and methionine (Met) in a 9:1 molar ratio, it was found that, surprisingly, 85 % of the proteins contained SeM residues, even though no SeM had been added, thus implying loss of the trifluoromethyl group from TFSeM. The transformation of TFSeM into SeM is enzymatically catalyzed by E. coli extracts, but TFSeM is not a substrate of E. coli methionine adenosyltransferase.

Identification of an intermediate methyl carrier in the radical S-adenosylmethionine methylthiotransferases RimO and MiaB.

RimO and MiaB are radical S-adenosylmethionine (SAM) enzymes that catalyze the attachment of methylthio (-SCH3) groups to macromolecular substrates. RimO attaches a methylthio group at C3 of aspartate 89 of protein S12, a component of the 30S subunit of the bacterial ribosome. MiaB attaches a methylthio group at C2 of N(6)-(isopentenyl)adenosine, found at nucleotide 37 in several prokaryotic tRNAs. These two enzymes are prototypical members of a subclass of radical SAM enzymes called methylthiotransferases (MTTases). It had been assumed that the sequence of steps in MTTase reactions involves initial sulfur insertion into the organic substrate followed by capping of the inserted sulfur atom with a SAM-derived methyl group. In this work, however, we show that both RimO and MiaB from Thermotoga maritima catalyze methyl transfer from SAM to an acid/base labile acceptor on the protein in the absence of their respective macromolecular substrates. Consistent with the assignment of the acceptor as an iron-sulfur cluster, denaturation of the SAM-treated protein with acid results in production of methanethiol. When RimO or MiaB is first incubated with SAM in the absence of substrate and reductant and then incubated with excess S-adenosyl-l-[methyl-d3]methionine in the presence of substrate and reductant, production of the unlabeled product precedes production of the deuterated product, showing that the methylated species is chemically and kinetically competent to be an intermediate.

A radically different mechanism for S-adenosylmethionine-dependent methyltransferases.

Methylation of small molecules and macromolecules is crucial in metabolism, cell signaling, and epigenetic programming and is most often achieved by S-adenosylmethionine (SAM)-dependent methyltransferases. Most employ an S(N)2 mechanism to methylate nucleophilic sites on their substrates, but recently, radical SAM enzymes have been identified that methylate carbon atoms that are not inherently nucleophilic via the intermediacy of a 5'-deoxyadenosyl 5'-radical. We have determined the mechanisms of two such reactions targeting the sp(2)-hybridized carbons at positions 2 and 8 of adenosine 2503 in 23S ribosomal RNA, catalyzed by RlmN and Cfr, respectively. In neither case is a methyl group transferred directly from SAM to the RNA; rather, both reactions proceed by a ping-pong mechanism involving intermediate methylation of a conserved cysteine residue.