The intestinal parasite Giardia lamblia
(alternatively G. intestinalis
) is a eukaryote belonging to the class Diplomonadida. This water-borne pathogen is prevalent worldwide and can be transmitted via
inadequately purified water or between infected humans. G. lamblia
is one of several pathogenic protozoa whose genomes are being mined by the MSGPP structural genomics collaboration in order to identify potential targets for the development of new antiparasitic drugs (Van Voorhis et al.
). Aminoacyl-tRNA synthetases (aaRSs) constitute one such class of potential drug targets, as they are key enzymes for protein synthesis in all organisms and hence, with rare exceptions, are essential for the growth or survival of the organism. Many eukaryotic genomes code for separate cytosolic and mitochondrial aaRS orthologs. However, G. lamblia
is notable both for the small size of its genome and for its lack of mitochondria, so unsurprisingly it contains only a single set of aaRS genes.
Each aaRS carries out two sequential reactions. It must recognize and activate the corresponding amino acid by attaching AMP, and it must specifically recognize and bind the cognate tRNA in order to transfer the activated amino acid to the terminal adenosine residue of the tRNA. In the case of prolyl-tRNA synthetase (ProRS) this corresponds to the two reactions (1) and (2),
Subsequently, the anticodon loop of the charged tRNA is matched by the ribosome to a complementary codon on an mRNA, leading to the incorporation of the amino acid that it carries into a growing protein chain. If specificity is lost at any of these three steps, i.e.
if the wrong amino acid is activated, if a noncognate tRNA is mistakenly charged or if the anticodon is paired with the wrong codon, then the result is the incorporation of an incorrect amino acid into a nascent protein.
Protein synthesis clearly depends on the existence of a well tuned pathway for the accurate activation and incorporation of each amino acid. Therefore, it is quite surprising that the genomes of some archaeal hyperthermophilic methanogens lack a recognizable gene coding for CysRS (Jacquin-Becker et al.
; Ruan et al.
). The missing essential functionality was at first attributed to the compensatory presence of a dual-specificity Pro/Cys-tRNA synthetase that was imputed to activate Cys and transfer it to tRNACys
as well as to activate Pro and transfer it to tRNAPro
(Lipman et al.
; Stathopoulos et al.
). Furthermore, the ProRS from G. lamblia
was initially reported to exhibit this same Pro/Cys dual specificity based on the observation that Cys was incorporated into bulk tRNA in the presence of G. lamblia
ProRS (Bunjun et al.
). However, it was later shown that misactivation of Cys is a general property of ProRS homologs from archaea, eukaryotes and some bacteria, but this activity is not accompanied by an ability to recognize or charge tRNACys
(Ahel et al.
; Ambrogelly et al.
). The previously reported incorporation of Cys into unfractionated tRNA in the presence of ProRS is adequately explained by the formation of misacylated Cys-tRNAPro
. Thus, despite its initial annotation as a dual-specificity aaRS, the G. lamblia
homolog for which the structure is reported here, functions biologically as a typical eukaryotic ProRS, albeit one with a surprisingly high off-target activity in activating Cys.