INVESTIGADORES
RAPISARDA Viviana Andrea
congresos y reuniones científicas
Título:
Mutations in the C-terminal region of NADH dehydrogenase-2 from Escherichia coli affect membrane anchoring
Autor/es:
VILLEGAS, J. M.; RINTOUL, M. R.; VOLENTINI, S. I.; RAPISARDA, V. A.
Lugar:
Villa Carlos Paz, Córdoba, Argentina
Reunión:
Congreso; VI Congreso de la Sociedad Argentina de Microbiología General (SAMIGE); 2009
Institución organizadora:
SAMIGE
Resumen:
Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane-bound flavoprotein, codified by ndh gene. It has been proposed that NDH-2 membrane anchorage could occur by multiple amphipatic turns along the enzyme. However, by bioinformatics studies performed in our laboratory, it has been identified a putative transmembrane C-terminal region. In order to validate whether this region could be responsible for NDH-2 membrane anchorage, several mutants were constructed, in which the enzyme C-terminal region was progressively deleted. They were called Trun-1 to Trun-4, lacking 13, 28, 43, and 57 aminoacids, respectively.  A NADH dehydrogenases deficient strain complemented with either the wild-type or the mutant ndh genes was used for all the experiments. For a preliminar screening of NDH-2 activity, an in vivo test was performed in minimal medium supplemented with mannitol as a sole carbon source at 30ºC, where it was described that NADH dehydrogenases null mutants are not able to grow. This phenotype was reverted only by the wild type and Trun-1 proteins. The rest of the truncated versions were not able to grow under these conditions. To check protein localization and enzymatic activities, the different cellular fractions were separated by ultracentrifugation. Trun-1 was localized in the membrane fraction as the wild type protein. The rest of the truncated forms were located only in cytosol, except for Trun-2 which was present in both fractions. NDH-2 enzymatic activities were measured, obtaining equivalent results to the in vivo experiment. Wild type and Trun-1 samples were active, while the rest of the truncated analogues showed no activity. However, with inactive mutants, activity was restored by the addition of FAD in the corresponding fractions. Taking together, we demonstrated that NDH-2 C-terminal region is implicated in the protein anchorage to the membrane. For the first time, we obtained an active and water-soluble NDH-2, which would be useful for detergent-free purification and further characterizations.