Small ubiquitin-like modifier (SUMO), a reversible post-translational protein 
modifier, plays important roles in diverse cellular mechanisms. Three enzymes, 
E1 (activating enzyme), E2 (conjugating enzyme) and E3 (ligase), are involved in 
SUMO modification. SUMOylation system and process in higher eukaryotes have been 
well studied. However, in protozoa, such as Trypanosoma brucei (T. brucei), 
these remain poorly understood. Herein, we identified the E1 (TbAos1/TbUba2) and 
E2 (TbUbc9) enzymes of SUMOylation pathway in T. brucei by sequence analysis and 
GST pull-down assay. Furthermore, we successfully reconstructed the SUMOylation 
system in vitro with recombinant enzymes. Using this system, the active site of 
TbUba2 and TbUbc9 was revealed to be located at Cys343 and Cys132, respectively, 
and a centrin homologue (TbCentrin3) was identified to be a target of 
SUMOylation in T. brucei. Altogether, our results demonstrate that TbAos1/TbUba2 
and TbUbc9 are the bona fide E1 and E2 enzymes of the SUMOylation system in T. 
brucei.