CONICET | Buscador de Institutos y Recursos Humanos

L-amino acids have been shown to be relevant in many Trypanosoma cruzi biologicalprocesses. However, little is known about the functions of D-amino acid metabolism on thebiology of the parasite. The proline racemase (PR) was firstly described in T. cruzi, as a potentmitogen. PR is also involved in the infectivity of the host cells by the parasite. The occurrence ofD-Alanine and an Alanine Racemase (AR) activity was also described in Leishmaniaamazonensis. We identified for the first time in pathogenic protozoa a gene encoding a putativeAR (TcAR), a pyridoxal 5?-phosphate (PLP) dependent enzyme that catalyzes the racemizationbetween L-and D-alanine. We cloned and expressed TcAR and characterized its recombinantproduct (rTcAR) of 43 kDa. Km and Vmax were determined for the reaction in both directions(21.5 ± 6.04 mM and 65 ± 9.4 μmol/min/mg (D to L) and 26.1 ± 7.9 mM and 53 ± 6.3μmol/min/mg (L to D)), with an optimal pH of 7-10 and an temperature of 25-37°C. Surprisingly,rTcAR was also able to racemize serine with a similar efficiency than alanine (Km : 30.74± 11.6 mM and Vmax : 51.2 ± 11.63 μmol/min/mg). Furthermore, contrary to bacterial AR, PLPseems not to be essential for the activity. Immunofluorescence with a specific anti-rTcARantibody showed a cytoplasmic localization for TcAR. Specific activity was measured along T.cruzi life cycle, with higher values in the insect stages when compared to mammalian stages.We demonstrated that, in addition to L-, D-alanine is also involved in osmoregulation.Importantly, only the L isoform can be a donor for transaminases. Interestingly, both D- and Lalaninewere able to maintain the parasites viability and intracellular ATP production.Nevertheless, only L-Alanine induced mitochondrial membrane protection and triggeredrespiration. Taken together our data show a functional TcAR and provide evidence of alaninebioenergetics functions in T. cruzi. Supported by:CNPq e FAPESP

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