INVESTIGADORES
RAPISARDA Viviana Andrea
congresos y reuniones científicas
Título:
A C-terminal single point mutation of Escherichia coli NADH dehydrogenase-2 affects enzymatic activity
Autor/es:
VILLEGAS, J. M.; ARCE, L. P.; RINTOUL, M. R.; RAPISARDA, V. A.
Reunión:
Congreso; VII CONGRESO DE LA SOCIEDAD ARGENTINA DE MICROBIOLOGÍA GENERAL (SAMIGE) Bicentenario; 2011
Resumen:
Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane-bound flavoprotein, encoded by ndh gene. In our laboratory, four NDH-2 mutants were constructed (named Trun-1 to Trun-4, lacking the last 13, 28, 43, and 57 aminoacids, respectively). When enzymatic activities were measured, it was observed that only Trun-1 was active similar to the wild type. The rest of the mutants restored part of the activity upon the addition of FAD 10 µM in the reaction mixture. In order to investigate this behaviour, we studied the region between Trun-1 and Trun-2 (from A406 to G420). Three single mutants were constructed, substituting H408, Y410, and K412 by alanine. These versions were then transformed into strain GNB10608 (nuo- ndh-), which lacks NADH dehydrogenase activities. As a rapid screening for NDH-2 activity, an in vivo test was performed in minimal medium supplemented with mannitol as a sole carbon source at 37ºC, where it was described that NADH dehydrogenases null mutants are not able to grow. This phenotype was reverted by wild type NDH-2, Y410xA and K412xA versions, while the mutant protein H408xA was not able to grow under this condition. Moreover, enzymatic activities were measured in membrane fractions. Y410xA and K412xA samples were active, whereas H408xA was less active in respect to the wild-type enzyme. However, in the last mutant, activity was restored by the addition of FAD in the mentioned fraction. Taking together, these results give us an idea that, within the region comprised by aminoacids 406 to 420, the H408 participates in NDH-2 activity, whether by a direct involvement in the FAD binding or by the production of a conformational change of the enzyme that may alters this union.