<p> The methylthiotransferase (MTTase) or miaB-like family is named after the (dimethylallyl)adenosine tRNA MTTase miaB protein, which catalyses a C-H to C-S bond conversion in the methylthiolation of tRNA. A related bacterial enzyme rimO performs a similar methylthiolation, but on a protein substrate. RimO acts on the ribosomal protein S12 and forms a separate MTTase subfamily. The miaB-subfamily includes mammalian CDK5 regulatory subunit-associated proteins and similar proteins in other eukaryotes. Two other subfamilies, yqeV and CDKAL1, are named after a Bacillus subtilis and a human protein, respectively. While yqeV-like proteins are found in bacteria, CDKAL1 subfamily members occur in eukaryotes and in archaebacteria. The likely MTTases from these 4 subfamilies contain an N-terminal MTTase domain, a central radical generating fold and a C-terminal TRAM domain (see <db_xref db="PROSITEDOC" dbkey="PDOC50926"/>). The core forms a radical SAM fold (or AdoMet radical), containing a cysteine motif CxxxCxxC that binds a [4Fe-4S] cluster [<cite idref="PUB00009728"/>, <cite idref="PUB00046148"/>, <cite idref="PUB00010539"/>]. A reducing equivalent from the [4Fe-4S]+ cluster is used to cleave S-adenosylmethionine (SAM) to generate methionine and a 5'-deoxyadenosyl radical. The latter is thought to produce a reactive substrate radical that is amenable to sulphur insertion [<cite idref="PUB00046148"/>, <cite idref="PUB00010539"/>]. The N-terminal MTTase domain contains 3 cysteines that bind a second [4Fe-4S] cluster, in addition to the radical-generating [4Fe-4S] cluster, which could be involved in the thiolation reaction. The C-terminal TRAM domain is not shared with other radical SAM proteins outside the MTTase family. The TRAM domain can bind to RNA substrate and seems to be important for substrate recognition. The tertiary structure of the central radical SAM fold has six beta/alpha motifs resembling a three-quarter TIM barrel core (see <db_xref db="PROSITEDOC" dbkey="PDOC00155"/>) [<cite idref="PUB00052321"/>]. The N-terminal MTTase domain might form an additional [beta/alpha]2 TIM barrel unit [<cite idref="PUB00046148"/>]. </p>