Dimerization and Threonine-Dependent Stabilization Govern Human YRDC Catalysis
Addition of N6-threonylcarbamoyladenosine occurs at position 37 (tA37) of five mitochondrial tRNA species, influencing translation fidelity and efficiency. Mutations in YRDC, the enzyme catalyzing the first step in tA37 synthesis, cause severe neurological and renal diseases in humans, including in Galloway-Mowatt disease (GAMOS). YRDC generates threonylcarbamoyl-AMP (TC-AMP) using ATP, threonine and bicarbonate as substrates, yet their binding order, residues involved in amino-acid selectivity, and precisely how mutations contribute to disease remain unclear. Here, we combine protein biophysics, mass spectrometry, NMR, mutagenesis and kinetics to define how the human enzyme operates. Differential scanning fluorimetry and circular dichroism identify L-threonine as the gatekeeper ligand. It binds with millimolar affinity, stabilizing YRDC and enhancing ATP binding nearly fortyfold, enabling bicarbonate association. Several amino acids can form aminoacylcarbamoyl-AMP adducts in vitro, but threonine yields superior protein stabilization and product formation. Native mass spectrometry and single-molecule mass photometry corroborate YRDCs dimeric state. GAMOS-linked mutations destabilize folding, disrupting dimerization and uncoupling substrate binding from catalysis, explaining functional losses. Supported by kinetic data and NMR, we propose a chemical mechanism for the reaction and identify dimerization and conformational stability as control points for activity. This work provides a foundation for designing selective YRDC inhibitors or enhancers.