Methylthiotransferase that catalyzes the conversion of N6-(dimethylallyl)adenosine (i(6)A) to 2-methylthio-N6-(dimethylallyl)adenosine (ms(2)i(6)A) at position 37 (adjacent to the 3'-end of the anticodon) of four mitochondrial DNA-encoded tRNAs (Ser(UCN), Phe, Tyr and Trp) (PubMed:22422838, PubMed:25738458, PubMed:28981754). Essential for efficient and highly accurate protein translation by the ribosome (PubMed:22422838, PubMed:25738458, PubMed:28981754). Specifically inhibits CDK5 activation by CDK5R1 (PubMed:11882646). Essential for efficient mitochondrial protein synthesis and respiratory chain; shows pathological consequences in mitochondrial disease (PubMed:25738458). [sulfur carrier]-SH + AH2 + N(6)-dimethylallyladenosine(37) in tRNA + 2 S-adenosyl-L-methionine = 2-methylsulfanyl-N(6)-dimethylallyladenosine(37) in tRNA + 5'-deoxyadenosine + [sulfur carrier]-H + A + 2 H(+) + L-methionine + S-adenosyl-L-homocysteine Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. Interacts with CDK5R1 (p35 form). CDK5RAP1, CDK5RAP2 and CDK5RAP3 show competitive binding to CDK5R1. Forms a complex with CDK5R1 and CDK5. Expressed in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (PubMed:10721722). Expressed in neurons of central nervous tissue (PubMed:10721722, PubMed:11882646). Mainly expressed in brain, placenta and testis. High expression in placenta and lung. May be due to intron retention. Absence of the mitochondrial target sequence which may lead to miss-localization. Belongs to the methylthiotransferase family. MiaB subfamily. CDK5RAP1 was proposed to act on both nuclear and mitochondrial RNA (PubMed:22422838). However, another study shows that ms2i6A is a mitochondrial tRNA specific modification and is absent from nuclear encoded RNA species, implying that there is no methylthiotransferase activity on nuclear RNA (PubMed:28981754). Truncated C-terminus.