Functional and structural investigation of N-terminal domain of the SpTad2/3 heterodimeric tRNA deaminase

Editing is a post-transcriptional process that changes the content of nucleic acids occurring on both DNA and RNA levels. Inosine at position 34 in tRNA is one such example, commonly produced via the deamination of A34, catalyzed by adenosine deaminase acting on tRNA (ADAT or Tad). The formation of inosine is essential for cell viability. The eukaryotic deaminases normally consist of the catalytic subunit Tad2 and the structural subunit Tad3, but the catalytic process is poorly understood. Despite the conservation of the (pseudo-) catalytic domains, the heterodimeric enzyme Tad2/3 also possesses additional domains that could exhibit novel functions. Here we present the structure of the N-terminal domain of the Schizosaccharomyces pombe Tad2/3 heterodimeric tRNA(A34) deaminase (N-SpTad2), which shares ~30% sequence identities with uridine-cytidine or pantothenate kinases, but lacks the predicted kinase functions. While biochemical assays indicated that the domain is not a nucleic-acid binder, it is able to significantly influence the A34-tRNA deamination activity of the holoenzyme. Through co-expression and purification analyses, we deduce that N-SpTad2 plays a role in mediating protein-protein contacts and enhancing the stability and solubility of SpTad2/3, without which the deaminase is not functional. Taken together, our structural and biochemical studies highlighted the importance of the additional domains to the intrinsic deaminase functions of heterodimeric Tad2/3 enzymes and promoted our understanding on this essential post-transcriptional tRNA modification.