Characterization of Trypanosoma brucei octanoyltransferase harnessing Bacillus subtilis mutants

SCATTOLINI A; LAVATELLI A; VACCHINA P; UTTARO AD; MANSILLA MC

Paraná

Congreso; LIV Reunión anual de la SAIB; 2018

SAIB

Lipoic acid (LA) is a universally conserved sulfur-containing cofactor involved in one-carbon and oxidative metabolism. Interfering with LAsynthesis would be a potential chemotherapeutic target against parasites like Trypanosoma cruzi and T. brucei, due to its essentiality to cellviability. However, while the ways by which proteins become lipoylated are very well characterized in prokaryotes, information concerningeukaryotes is scarce. It was previously found by in silico analyses that T. brucei Tb11.01.1160 gene product (TbLipT) is similar to bacterial andyeast octanoyltransferases. It was also demonstrated that it functionally complements a lip2 mutant in Saccharomyces cerevisiae. These resultsstrongly suggested that TbLipT is the T. bruceioctanoyltransferase. In order to deeply characterize the substrate specificity of the octanoytransferreaction we expressed this protein in B. subtilis mutants defective in different steps of the lipoylation pathway, and observed the functionalcomplementation and protein lipoylation patterns of the transformed mutants. The fact that the B. subtilisΔgcvH and ΔlipL mutants were notcomplemented, suggests that TbLipT transfers octanoate specifically to the H protein, in a similar way as LipM in B. subtilis and Lip2 in yeasts.By site-directed mutagenesis it could also be confirmed the importance of Lys161 and Cys195 for the octanoyltransferase activity of thetrypanosomal protein.