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.

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.

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.