CONICET | Buscador de Institutos y Recursos Humanos

Functional characterization of the D-3-phosphoglycerate dehydrogenase in L. mexicana. Giordana Lucila, Nowicki Cristina. Fac. de Farmacia y Bioquímica, UBA-CONICET, Junín 956, Bs As, Argentina. Leishmania parasites are the causative agents of a spectrum of neglected diseases. The current clinical treatments are far from being satisfactory, the available drugs are highly toxic and of low efficacy. These pathogens exhibit unusual metabolic features; therefore, the understanding of their biochemical peculiarities is of great interest in public health. During their life cycles, these pathogens have to adapt to changing microenvironments, which also are highly dissimilar in regards to the availability of nutrients. It is accepted that glucose is scarce in phagolysosomes, and therefore, amastigotes could depend on amino acids as carbon and energy sources. Besides, proteomic analyses of the in vitro induced differentiation stages support the idea that certain amino acids, such as serine and cysteine among others could be biosynthesized in amastigotes. In most organisms the D-3-phosphoglycerate dehydrogenase (PGDH) is involved in L-Ser production. This enzyme could be linked to NAD or NADP and catalyzes the oxidation of 3-phosphoglycerate into 3-phosphohydroxypyruvate (3-PHP). PGDH is comprised within the pathway referred as phosphorylated route; in which a phosphoserine aminotransferase (PSAT) transforms 3-PHP into 3-phosphoserine and ultimately a phosphatase leads to L-Ser production. Putative PGDHs and PSATs are conserved in the genomes of Leishmania parasites but not in those of trypanosomes. Previous reports showed that cell-free extracts of L. mexicana promastigotes exhibited PGDH activity, being the latter augmented in amastigotes. However, neither PGDH nor PSAT has been functionally characterized yet. Therefore, we have embarked on the biochemical characterization of L. mexicana PGDH and PSAT by cloning the putative genes and expressing the recombinant proteins in Escherichia coli. Herein, we present the results corresponding to the partial characterization of L. mexicana PGDH. Sequence analysis of the putative PGDH put in evidence a remarkable similarity with bacterial homologues as well as protozoal counterparts such as that of Entamoeba histolytica. To facilitate the purification of the recombinant protein, the N-terminus of L. mexicana PGDH was extended by a 6xHis-tag. Upon affinity chromatography, the enzyme exhibited protein homogeneity when subjected to SDS-PAGE, displayed a specific activity of about 10 U.mg-¹ and showed to utilize specifically NAD as coenzyme. Unlike the bacterial homologues, the L. mexicana enzyme seemed not to be allosterically inhibited by the end product of the route, L-serine.