RESUMO

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 ∼5-10 °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.

Assuntos

Proteínas de Bactérias/química , Escherichia coli/enzimologia , NADH Desidrogenase/química , Proteínas de Bactérias/genética , Dicroísmo Circular , Estabilidade Enzimática , Escherichia coli/química , Flavina-Adenina Dinucleotídeo/química , Cinética , NADH Desidrogenase/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura