RimO, a MiaB-like enzyme, methylthiolates the universally conserved Asp88 residue of ribosomal protein S12 in Escherichia coli

Abstract

Ribosomal protein S12 undergoes a unique posttranslational modification, methylthiolation of residue D88, in Escherichia coli and several other bacteria. Using mass spectrometry, we have identified the enzyme responsible for this modification in E. coli, the yliG gene product. This enzyme, which we propose be called RimO, is a radical-S-adenosylmethionine protein that bears strong sequence similarity to MiaB, which methylthiolates tRNA. We show that RimO and MiaB represent two of four subgroups of a larger, ancient family of likely methylthiotransferases, the other two of which are typified by Bacillus subtilis YqeV and Methanococcus jannaschii Mj0867, and we predict that RimO is unique among these subgroups in its modification of protein as opposed to tRNA. Despite this, RimO has not significantly diverged from the other three subgroups at the sequence level even within the C-terminal TRAM domain, which in the methyltransferase RumA is known to bind the RNA substrate and which we presume to be responsible for substrate binding and recognition in all four subgroups of methylthiotransferases. To our knowledge, RimO and MiaB represent the most extreme known case of resemblance between enzymes modifying protein and nucleic acid. The initial results presented here constitute a bioinformatics-driven prediction with preliminary experimental validation that should serve as the starting point for several interesting lines of further inquiry.

Fig. 1.

Schematic structures of the five known naturally occurring methylthiol residues from proteins and nucleic acids. Methylthiol moieties are shown in red. R, ribose. (A) β-methylthio-aspartic acid. (B) ms²i⁶A when X = H and ms²io⁶A when X = OH. (C) ms²t⁶A when Y = H and ms²hn⁶A when Y = CH₃.

Fig. 2.

Deconvoluted ESI-TOF MS of ribosomal protein S12 from various E. coli strains. S12 peaks are indicated by asterisks (*). (A) Protein from wild-type strain MG1655. (B) Protein from mutant strain FB23662 (yliG⁻). (C) Protein from mutant strain ER3051/pLIT-yliG.

Fig. 3.

Multiple sequence alignment of eight diverse MTTases (two from each of the four subfamilies): RimO_Eco (YliG/RimO from E. coli K-12; NP_415356), RimO_Aae (aq_849 from Aquifex aeolicus; NP_213577), MiaB_Eco (MiaB from E. coli K-12; NP_415194), MiaB_Aae (aq_284 from A. aeolicus; NP_213198), YqeV_Bsu (BSU25430/YqeV from B. subtilis; NP_390421), YqeV_Aae (aq_474 from A. aeolicus; NP_213332), Mj08_Mja (Mj0867 from M. jannaschii; NP_247862), Mj08_Hsa (CDKAL1 from Homo sapiens; NP_060244). The three domains as defined by Pfam are boxed, inclusive of edges: UPF0004 (pink), radical-SAM (cyan), and TRAM (brown). Residues conserved in the larger alignment, listed in SI Table 2, are boxed in black, and additional residues conserved in all eight sequences in this alignment are boxed in gray. (For this purpose, the following residues are treated as equivalent: S = T, E = D, K = R, F = W = Y, and I = L = V.) Subfamily-specific residues as defined in SI Table 3 are boxed in the color of the subfamily that differs from the remaining three: blue for RimO, red for MiaB, green for YqeV, and yellow for Mj0867. The three cysteines that coordinate the [4Fe-4S] cluster in the radical-SAM domain of MiaB are indicated by *, the three cysteines that coordinate the [4Fe-4S] cluster in the UPF0004 domain of MiaB are indicated by §, and residues that may be involved with SAM binding (see SI Table 2) are indicated by †. The NPPY motif conserved among eukaryotic Mj0867 members is boxed in yellow. At the lower right is a schematic phylogenetic tree, based on SI Fig. 5, showing the relationships of these sequences to one another.