The human tRNA taurine modification enzyme GTPBP3 is an active GTPase linked to mitochondrial diseases

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• GTPBP3 and MTO1 are essential for 5-taurinomethyluridine (τm5U) biosynthesis in mitochondrial tRNAs.
• Defects in τm5U modification are linked to various human diseases.
• Previous studies faced challenges in reconstituting and mechanistically studying τm5U modification due to enzyme purity and activity issues.

Purpose of the Study:

• To identify the active form of human GTPBP3 (hGTPBP3) and characterize its GTPase activity.
• To investigate the mechanism of GTP hydrolysis by hGTPBP3.
• To explore the functional and structural impact of pathogenic hGTPBP3 mutations and discover novel isoforms.

Main Methods:

• Purification and characterization of mature hGTPBP3.
• In vitro GTPase assays using wild-type and mutant hGTPBP3.
• Analysis of hGTPBP3 localization and function in vivo.
• Identification and characterization of a novel cytoplasmic hGTPBP3 isoform.

Main Results:

• The mature form of hGTPBP3 was identified and demonstrated to be an active GTPase in vitro, essential for tRNA modification in vivo.
• The isolated G domain and N-terminally truncated mutants showed limited GTP hydrolysis activity with high Km values.
• Pathogenic mutations in hGTPBP3 were linked to altered enzyme localization, structure, and function.
• A novel cytoplasm-localized hGTPBP3 isoform with potential noncanonical functions was discovered.

Conclusions:

• The GTP hydrolysis mechanism of hGTPBP3 has been elucidated for the first time.
• This study provides a foundation for understanding the τm5U modification pathway and the etiology of related diseases.
• The discovery of a novel isoform suggests unexplored functions of hGTPBP3 beyond mitochondrial tRNA modification.