Catalytic and structural characterisation of human YRDC and enzymatic activation of ProTide prodrugs

N⁶-threonylcarbamoyladenosine (t⁶A₃₇) is a conserved tRNA modification that stabilises ANN codon decoding and supports translational fidelity. YRDC catalyses the first step of t⁶A₃₇ biosynthesis and has been linked to severe neurodevelopmental and renal disease, including Galloway–Mowat syndrome (GAMOS). Despite its biological importance, key features of human YRDC remained unclear, including substrate-binding order, amino-acid selectivity, oligomeric state, and the molecular basis of disease-associated dysfunction. In the first part (Chapters 1–5), human YRDC was characterised biochemically and structurally. Recombinant YRDC was shown to catalyse the formation of threonylcarbamoyl-AMP (TC-AMP) intermediate from ATP, L-threonine, and bicarbonate. TC-AMP decay was quantified, allowing AMP formation to be used as a reliable kinetic readout. Binding and steady-state analyses identified L-threonine as the gatekeeper ligand, as it stabilised a more ordered protein state, strengthened ATP binding, and enabled detectable bicarbonate binding. Related amino acids bound weaker and supported slower formation of TC-like products, showing that YRDC is modestly promiscuous in vitro but remains functionally preference for L-threonine. Quaternary-structure analysis further showed that wild-type human YRDC is predominantly dimeric in solution. Active-site substitutions impaired catalysis while largely preserving a folded core, whereas three GAMOS-associated variants destabilised the protein, increased disorder, and shifted the equilibrium away from the native dimer towards monomeric or aberrant oligomeric species. The second part of this thesis (Chapters 6–8) examined enzymatic activation of nucleotide ProTides, a prodrug platform designed to improve the pharmacological properties of nucleoside analogues. Using the cordycepin ProTide NUC-7738 and the 5-fluorouracil ProTide NUC-3373 as model compounds, hHint1 was cloned, ex-pressed, and purified, and selected esterases, including neutrophil elastase, CES1, and CES2, were assessed as candidate activating enzymes. Esterase assays with two NUC-3373 diastereoisomers showed only modest, statistically non-significant differences in product formation. Together, these results define a mechanistic framework for YRDC function and establish an experimental basis for future studies of ProTide activation and optimisation.