Biophysical and biochemical characterization of human tRNA nucleotidyltransferase variants

The enzyme ATP(CTP):tRNA nucleotidyltransferase (tRNA-NT) is required for tRNA maturation and repair. This enzyme adds to tRNAs the universally conserved 3’-cytidine-cytidine-adenosine (CCA) sequence required for aminoacylation. Given the essential role of this enzyme in protein synthesis, human disease phenotypes have been linked to mutations in the gene encoding this protein (Aksentijevich et al. 2014; Chakraborty et al. 2014; Sasarman et al. 2015; DeLuca et al. 2016; Hull et al. 2016; Wedatilake et al. 2016; Frans et al. 2017; Lougaris et al. 2018; Giannelou et al. 2018; Bader-Meunier et al. 2018; Gorodetsky et al. 2018; Kumaki et al. 2019; Abdulhadi et al. 2019). The variant proteins characterized to date have shown reduced thermostability relative to the native enzyme (Leibovitch et al. 2018, 2019), suggesting that reduced stability of tRNA-NT may lead to the phenotypes observed. Here, we looked at additional variant proteins to see if these disease-linked variants also show reduced thermostability. The variant proteins characterized here were E43Δ which had a glutamate residue deleted near the N-terminus of the protein, R99W which contained an arginine-to-tryptophan substitution near the active site of the enzyme, and A[8] with a frame shift that truncated the protein by nine amino acids and altered the new C-terminal eight amino acids. Interestingly, biophysical and biochemical characterization of these variants showed no major decrease in thermostability relative to the native enzyme. However, all three variants showed a reduced ability to incorporate AMP into a specific tRNA template in vitro. This suggests that, in addition to reduced thermostability, effects in AMP incorporation may also be linked to a disease phenotype. Interestingly, the E43Δ, R99W and A[8] variants all affected different aspects of AMP incorporation, reflecting their different locations in the protein and suggesting that AMP incorporation may be mediated by different domains and substructures spanning the entire enzyme and not confined simply to the active site.