Amidotransferase activity as a target for chemoterapeutic development against Trypanosoma brucei

SCATTOLINI, A; MANSILLA, MC; UTTARO, A

Congreso; Congreso Conjunto SAIB-SAMIGE 2021; 2021

Lipoic acid (LA) is a universally conserved sulfur-containing cofactor involved in one-carbon and oxidative metabolism. LA can be acquired by a salvage pathway, in which it is attached to their cognate enzymes by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as ?lipoyl-relay?, requires two additional proteins, GcvH, the glycine cleavage system H subunit, and the amidotransferase, LipL. Interfering LA synthesis would be a potential chemotherapeutic target against parasites like Trypanosoma cruzi and T. brucei, due to the essentiality of protein lipoylation for cell viability. By complementation of different mutants of Bacillus subtilis we identified TbLipL as the amidotransferase of the parasite. This protein shares most of its N-terminal amino acid sequence with bacterial amidotransferases but it has an additional C-terminal domain. Primary structure of this domain is highly conserved in Trypanosomas but differs from those of other eukaryotes. We found that the truncated version of TbLipL, lacking this C-terminal domain, was unable to restore growth of a mutant strain of B. subtilis deficient in amidotransferase activity, indicating that it is essential either for catalysis or proper folding. It is remarkable that TbLipL lacks a cysteine residue equivalent to C150 of B. subtilis, identified as essential for the amidotransfer reaction, and conserved in bacterial proteins. This seems to be a common characteristic of eukaryotic amidotransferases, which only share the conserved lysine present in the biotin/lipoyl protein ligase family. The essentiality of the conserved lysine residue (Lys169) in trypanosomal amidotransferase activity was assessed by site-directed mutagenesis, suggesting that the reaction mechanisms of bacterial and eukaryotic enzymes would be different. It was described that treatment with Bromooctanoate (BrO) inhibited the growth of epimastigote forms of T. cruzi as a consequence of lack in E2 lipoylation. Taking advantage that BrO had no inhibitory effects on B. subtilis, we demonstrated that this compound specifically inhibits TbLipL activity.Our results show for the first time the presence of a lipoyl-relay pathway in a parasitic protozoan, expanding to Excavata the range of organisms having this kind of metabolism, and positioning the trypanosomatid amidotransferase as a valid target for drug intervention.