CDK5RAP1 (CDK5RAP1 mitochondrial tRNA methylthiotransferase) functions as a radical S-adenosylmethionine enzyme that catalyzes the 2-methylthio (ms²) modification of four mitochondrial tRNAs (Ser(UCN), Phe, Tyr, and Trp) at position 37 adjacent to the anticodon 1. This post-transcriptional modification is essential for maintaining mitochondrial protein synthesis fidelity and respiratory chain function 1. The enzyme converts N6-(dimethylallyl)adenosine to 2-methylthio-N6-(dimethylallyl)adenosine, ensuring accurate protein translation by mitochondrial ribosomes 1. CDK5RAP1 deficiency severely impairs mitochondrial protein synthesis, leading to respiratory defects and making cells susceptible to stress-induced mitochondrial dysfunction 1. In disease contexts, CDK5RAP1 deficiency induces apoptosis in cancer cells through ROS/JNK and NF-κB signaling pathways, suggesting potential therapeutic applications 23. The ms² modifications are sensitive to oxidative stress and reduced in mitochondrial disease patients, highlighting their pathological significance 1. Multiple splice variants exist with tissue-specific expression patterns, particularly in brain, placenta, and testis 4. CDK5RAP1 represents one of only eight radical SAM enzymes in humans and is critical for mitochondrial function and cellular energy metabolism 5.