NADH oxidase ofThermus thermophilus HB8 overproduced fromEscherichia coli

Summary An NADH oxidase purified from the extreme thermophileThermus thermophilus HB8 is a monomeric flavoprotein with a 1 1 ratio of flavin-adenine dinucleotide (FAD) to the polypeptide chain. It catalyzes in vitro the oxidation of reduced NADH or NADPH with the formation of H₂O₂. The gene encoding the NADH oxidase fromT. thermophilus HB8 was cloned, and its nucleotide sequence was determined. The molecular mass of 22,749 Da, as deduced from thenox gene, agrees with that of the purified NADH oxidase fromT. thermophilus HB8, as estimated by mass spectrometry. Thenox gene does not contain a GX₄GK consensus sequence typical for nucleotide binding proteins. Thenox gene was overexpressed inEscherichia coli, and a protocol for the rapid purification of theE. coli-borneT. thermophilus NADH oxidase or its His₆-tagged analogue was developed by using thermal denaturation step and affinity chromatography.