The methylthiotransferase (MTTase) or miaB-like family is named after the (dimethylallyl)adenosine tRNA MTTase miaB protein, which catalyzes 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 a N-terminal MTTase domain, a central radical generating fold and a C-terminal TRAM domain (see <PDOC50926>). The core forms a radical SAM fold (or AdoMet radical), containing a cysteine motif CxxxCxxC that binds a [4Fe-4S] cluster [1,2,3,4]. 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 sulfur insertion [3,4]. 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 β/α motifs resembling a three-quarter TIM barrel core (see <PDOC00155>) [5]. The N-terminal MTTase domain might form an additional [β/α]2 TIM barrel unit [3].

Some proteins known to contain a MTTase domain are listed below:

• Bacterial (Dimethylallyl)adenosine tRNA methylthiotransferase miaB (yleA), which catalyzes the methylthiolation of N6-(dimethylallyl)adenosine (i6A), leading to the formation of 2-methylthio-N6-(dimethylallyl)adenosine (ms2i6A) at position 37 in tRNAs that read codons beginning with uridine.
• Human CDK5 regulatory subunit-associated protein 1 (CDK5RAP1), which specifically inhibits CDK5 activation by CDK5R1.
• Plant CDK5RAP1-like protein, a miaB-like potential regulator of CDK5 activity.
• Caenorhabditis elegans and amoebal CDK5RAP1-like proteins, miaB-like potential regulators of CDK5 activity.
• Escherichia coli ribosomal protein S12 methylthiotransferase rimO (yliG), which catalyzes the methylthiolation of the residue Asp-88 of ribosomal protein S12.
• Synechocystis strain PCC 6803 ribosomal protein S12 methylthiotransferase rimO (slr0082).
• Bacillus subtilis putative methylthiotransferase yqeV (EC 2.-.-.-).
• Mycoplasma iowae putative methylthiotransferase in 16S RNA 5'region.
• Helicobacter pylori putative methylthiotransferase HP_0285 (EC 2.-.-.-).
• Rickettsia prowazekii putative methylthiotransferase RP416 (EC 2.-.-.-).
• Vertebrate CDK5 regulatory subunit-associated protein 1-like 1 (CDKAL1).
• Fruit fly CDKAL1-like protein (CG6550).
• Caenorhabditis elegans CDKAL1-like protein (Y92H12BL.1).
• Methanocaldococcus jannaschii CDKAL1-like putative methylthiotransferases MTH_826 and MJ0867 (EC 2.-.-.-).
• Methanocaldococcus jannaschii putative methylthiotransferase MJ0865.

These proteins range in size from about 47 to 61 Kd. They contain six conserved cysteines, three of which form the motif in the central radical SAM fold that can be used as a signature pattern [1]. We also developed a profile that covers the N-terminal MTTase domain, including the other [4Fe-4S] cluster binding cysteines.