Methylthiotransferases are enzymes of the radical S-adenosyl methionine (radical SAM) superfamily. These enzymes catalyze the addition of a methylthio group to various biochemical compounds including tRNA and proteins. Methylthiotransferases are classified into one of four classes based on their substrates and mechanisms. All methylthiotransferases have been shown to contain two Fe-S clusters, one canonical cluster and one auxiliary cluster, that both function in the addition of the methylthio group to the substrate.