Characterization of Trypanosoma brucei octanoyltransferase harnessing Bacillus subtilis mutants

SCATTOLINI ALBERTINA; UTTARO ANTONIO; LAVATELLI ANTONELA; MANSILLA MARÍA CECILIA; VACCHINA PAOLA

Paraná

Congreso; LIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

Lipoic acid (LA) is a universally conserved sulfur-containing cofactor involved in one-carbon and oxidative metabolism. Interfering with LA synthesis would be a potential chemotherapeutic target against parasites like Trypanosoma cruzi and T. brucei, due to its essentiality to cell viability. However, while the ways by which proteins become lipoylated are very well characterized in prokaryotes, information concerning eukaryotes is scarce. It was previously found by in silico analyses that T. brucei Tb11.01.1160 gene product (TbLipT) is similar to bacterial and yeast octanoyltransferases. It was also demonstrated that it functionally complements a lip2 mutant in S. cerevisiae. These results strongly suggested that TbLipT is the T. brucei octanoyltransferase. In order to deeply characterize the substrate specificity of the octanoytransfer reaction we expressed this protein in B. subtilis mutants defective in different steps of the lipoylation pathway, and observed the functional complementation and protein lipoylation patterns of the transformed mutants. The fact that the B. subtilis ΔgcvH and ΔlipL mutants were not complemented, 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 the trypanosomal protein.