Maintenance and Thermal Stabilization of HADH dehydrogenase-2 conformation upon elimination of its C-Terminal región

VILLEGAS JM.,; TORRES-BUGEAU CM.,; CHEÍN R.,; BURGOS I.,; FIDELIO G.D.,; RINTOUL M.R.,; RAPISARDA VA.

TUCUMAN, ARGENTINA

Congreso; XLI REUNION NACIONAL DE LA SOCIEDAD ARGENTINA DE BIOFISICA; 2012

SOC ARG DE BIOFISICA

Development of an artificial enzyme with activity and structure comparable to that of natural enzymes is an important goal in biological chemistry. Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein, belonging to a group of enzymes with scarce structural information. By eliminating the C-terminal region of NDH-2, a water soluble version with significant enzymatic activity was previously obtained. Here, NDH-2 structural features were established, in comparison to those of the truncated version. Far-UV circular dichroism, Fourier transform infrared spectroscopy and limited proteolysis analysis showed that the overall structure of both proteins was similar at 30 C. Experimental data agree with the predicted NDH-2 structure (PDB: 1OZK). The absence of C-terminal region stabilized in w5e10 C the truncated protein conformation. However, truncation impaired enzymatic activity at low temperatures, probably due to the weak interaction of the mutant protein with FAD cofactor.