Identification of an Intermediate Methyl Carrier in the Radical S-Adenosylmethionine Methylthiotransferases RimO and MiaB

Abstract

RimO and MiaB are radical S-adenosylmethionine (SAM) enzymes that catalyze the attachment of methylthio (–SCH3) groups onto 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 N6-(isopentenyl)adenosine, found at nucleotide 37 in several prokaryotic tRNAs. These two enzymes are prototypical members of a subclass of radical SAM (RS) 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 (Tm) 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 (Fe/S) 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 ([methyl-d3]-SAM) 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.

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

“…It may be that a similar reassembly process is required for the [4Fe-4S] auxiliary cluster of lipoyl synthase, but this remains experimentally untested. The proposed sacrificial role of the auxiliary clusters of BioB and LipA contrasts significantly with the proposed mechanism of the methylthiotransferases MiaB [24,25] and RimO [22,23,26]. The structure of holoRimO [27] includes a pentasulfide (S 5 2-) ligand bridging between two [4Fe-4S] clusters separated by only 8 Å.…”

“…The biochemical and structural data for MiaB led to a proposed mechanism in which an exogenous sulfur donor (such as MeS -) is activated by binding to the auxiliary cluster at an otherwise free binding site (i.e. without a permanent protein ligand in the resting state) [26]. The multiple turnovers of both RimO and MiaB with CH 3 Se -or CH 3 Ssubstrates support this hypothesis [27].…”

“…The high degree of sequence similarity between biotin synthase and lipoyl synthase [15] which are both required for transformations resulting in sulfur insertion [17] suggests they may function by related mechanisms and kinetic [18], structural [19] and spectroscopic data [20,21] indicate that the auxiliary [2Fe-2S] cluster of BioB functions is the sulfur donor for biotin formation. A further subgroup of the RS superfamily, the methylthiotransferases, catalyze the insertion of thiomethyl groups into C-H bonds of proteins [22,23] and nucleic acids [24][25][26], but these enzymes can use exogenously added sulfide as a sulfur source [27]. N Ɛ -octanoyl lysine containing peptides have been used as model substrates for SsLipA to yield lipoyl peptides as products [28].…”

SPASM and Twitch Domains in S-Adenosylmethionine (SAM) Radical Enzymes

“…Methylthioltransferases (MTTases), as their name suggests, transfer a methylthiol group to macromolecular substrates including the S12 ribosomal protein (catalyzed by RimO) and tRNA-A37 (catalyzed by MiaB). Methylthioltransferases also have auxiliary clusters (52,53), which, unlike for BioB and lipoyl synthase, are not currently believed to serve as the source of sulfur for generation of the methylthiol group (54). Instead, a recent crystal structure of RimO has led to the proposal that its auxiliary cluster is responsible for binding a polysulfide moiety that serves as the sulfur source (55).…”

Discovery of Multiple Modified F 430 Coenzymes in Methanogens and Anaerobic Methanotrophic Archaea Suggests Possible New Roles for F 430 in Nature

“…MTTases, exemplified by MiaB and RimO, catalyze the generation of an -SCH 3 group in place of a hydrogen on either tRNA or a ribosomal protein substrate, respectively, resulting in the formation of a methyl thioether (47). The source of the sulfur for this reaction is still unclear, but it has been proposed to arise from a sulfide coordinated by an auxiliary [4Fe-4S] cluster (cluster II) (48,49). The methyl group is derived from one of the two SAM molecules required for the reaction (47).…”