This study focuses on the adenosine deaminase that acts on tRNA (ADAT) at the wobble position found in Trypanosoma brucei. This essential enzyme facilitates the deamination of adenosine (A) to inosine (I) resulting in an edited tRNA that has the ability to recognize three unique codons. In addition we discovered a unique cytidine (C) to uridine (U) editing event in tRNAThr(AGU) at position 32. Our discovery that C to U editing at position 32 stimulates A to I editing at position 34 indicated an interplay between the two editing sites which supports our previously proposed interdependence model for RNA editing. The observation that C to U editing occurs in cytoplasmic tRNAs indicated that editing in tRNAs is more widespread that previously thought and is not restricted to organellar tRNAs.
Recombinant co-expression of TbADAT2 and TbADAT3 resulted in an enzyme that is catalytically similar to the native enzyme. Site directed mutagenesis was used to determine the role of various domains of TbADAT2 and TbADAT3 in the deamination reaction. Our data show that both TbADAT2 and TbADAT3 play critical roles in catalysis in contrast to similar experiments conducted in yeast showing that mutations to conserved residues of ADAT3 had little effect on catalysis. Our results also indicate that the C-terminus of TbADAT2 contains critical residues that are involved in catalysis and tRNA binding. Footprint analyses suggests that this portion of the enzyme contacts the D and TYC arm of the tRNA. We speculate that by contacting the tRNA substrates with a string of charged residues at the C-terminus of the enzyme, the deaminase uses this general RNA binding domain to interact with additional substrates, different than the bacterial deaminases which only recognize one substrate, tRNAArg. In this fashion, the T. brucei ADAT enzyme has the ability to accommodate the eight different tRNAs that must be deaminated in trypanosomes.
Although a great deal of work has gone into dissecting the bacterial ADATs but little emphasis has been directed toward eukaryotic ADATs. This study provides now insight into the catalytic and substrate recognition differences between bacterial and eukaryotic ADATs.