Student Theses and Dissertations

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RU Laboratory

Stebbins Laboratory


tRNA is edited at the wobble position of the anticodon to accommodate the degeneracy of the genetic code. The enzymes responsible for deamination of adenosine 34 to inosine are found in all organisms and in all but a few cases are essential for life. In bacteria the reaction is carried out by the homodimeric adenosine deaminase acting on tRNA (ADAT) and modifies only tRNAarg, whereas in eukarya the enzyme is the ADAT2/3 heterodimer, which performs the same reaction on all tRNA molecules with adenosine at position 34. Despite a similar evolutionary origin, the characteristics of these two enzymes are significantly different. Substrate binding and catalysis occur within the active site of the bacterial homologue, which can act on both stem-loop substrate analogues and full-length tRNA. Eukaryotic heterodimers function differently with separate substrate recognition and enzymatic components and are only active on full-length tRNA. Furthermore, in trypanosomatids, the ADAT2/3 complex has been shown to also exhibit C-to-U deamination in vitro, and the ADAT2 protein alone has been shown to play a role in Cto- U editing in vivo at the C32 position of tRNA. The basis for these extra enzymatic activities is unknown. ADAT2 homodimers from trypanosomes and humans have been shown to be stable in vitro but these homodimers show dramatically decreased tRNA binding and have no detectable enzymatic activity toward the typical tRNA substrates. While crystallographic data exist for the human ADAT2, the structural underpinnings for both the lack of canonical enzymatic activity and the acquisition of additional functionality in the trypanosomatid enzymes remain unanswered. In the presented work xray crystallography was used to determine the structure of ADAT2 from T. brucei. The solution of the structure was technical, requiring several different crystallographic techniques at each step of the process. These are described in detail. The final structure revealed an enzyme harboring the canonical cytidine deaminase fold and an otherwise normal functional-appearance with no obvious indications as to why this enzyme is inactive. Potential reasons for the loss of substrate binding were indicated by mutations in the regions of ADAT2 analogous to the bacterial enzyme. Further analysis identified three loop regions that are unique to trypanosomatid ADAT2 and are located in regions of structural significance so as to suggest a mechanism for an altered substrate profile for this orphan enzyme.


A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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