A C-terminal single point mutation in Escherichia coli NADH dehydrogenase-2 affects enzymatic activity

JOSEFINA M. VILLEGAS, LORENA P. ARCE, MARÍA R. RINTOUL, VIVIANA A. RAPISARDA

San Miguel de Tucuman

Congreso; VII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE DEL BICENTENARIO "Dedicado a la presentación de trabajos de investigación básica sobre microorganismos; 2011

Sociedad Argentina de Microbiologia General SAMIGE

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 10 μM FAD 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 induction of a conformational change.