Analysis of the pathogenic I326T variant of human tRNA nucleotidyltransferase reveals reduced catalytic activity and thermal stability in vitro linked to a conformational change.

Biochim Biophys Acta Proteins Proteom 2019 Jun 5;1867(6):616-626. Epub 2019 Apr 5.

Department of Chemistry and Biochemistry and Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke St. W., Montréal H4B 1R6, Québec, Canada. Electronic address:

The I326T mutation in the TRNT1 gene encoding human tRNA nucleotidyltransferase (tRNA-NT) is linked to a relatively mild form of SIFD. Previous work indicated that the I326T variant was unable to incorporate AMP into tRNAs in vitro, however, expression of the mutant allele from a strong heterologous promoter supported in vivo CCA addition to both cytosolic and mitochondrial tRNAs in a yeast strain lacking tRNA-NT. To address this discrepancy, we determined the biochemical and biophysical characteristics of the I326T variant enzyme and the related variant, I326A. Our in vitro analysis revealed that the I326T substitution decreases the thermal stability of the enzyme and causes a ten-fold reduction in enzyme activity. We propose that the structural changes in the I326T variant that lead to these altered parameters result from a rearrangement of helices within the body domain of the protein which can be probed by the inability of the monomeric enzyme to form a covalent dimer in vitro mediated by C373. In addition, we confirm that the effects of the I326T or I326A substitutions are relatively mild in vivo by demonstrating that the mutant alleles support both mitochondrial and cytosolic CCA-addition in yeast.

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http://dx.doi.org/10.1016/j.bbapap.2019.04.003DOI Listing
June 2019

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