The Human Trna Taurine Modification Enzyme Gtpbp3 Is An Active Gtpase Linked To Mitochondrial Diseases

GTPBP3 and MTO1 cooperatively catalyze 5-taurinomethyluridine (tau m(5)U) biosynthesis at the 34(th) wobble position of mitochondrial tRNAs. Mutations in tRNAs, GTPBP3 or MTO1, causing tau m(5)U hypomodification, lead to various diseases. However, efficient in vitro reconstitution and mechanistic study of tau m(5)U modification have been challenging, in part due to the lack of pure and active enzymes. A previous study reported that purified human GTPBP3 (hGTPBP3) is inactive in GTP hydrolysis. Here, we identified the mature form of hGTPBP3 and showed that hGTPBP3 is an active GTPase in vitro that is critical for tRNA modification in vivo. Unexpectedly, the isolated G domain and a mutant with the N-terminal domain truncated catalyzed GTP hydrolysis to only a limited extent, exhibiting high K-m values compared with that of the mature enzyme. We further described several important pathogenic mutations of hGTPBP3, associated with alterations in hGTPBP3 localization, structure and/or function in vitro and in vivo. Moreover, we discovered a novel cytoplasm-localized isoform of hGTPBP3, indicating an unknown potential noncanonical function of hGTPBP3. Together, our findings established, for the first time, the GTP hydrolysis mechanism of hGTPBP3 and laid a solid foundation for clarifying the tau m(5)U modification mechanism and etiology of tau m(5)U deficiency-related diseases.