NADH OXIDASE OF THERMUS-THERMOPHILUS HB8 OVERPRODUCED FROM ESCHERICHIA-COLI

An NADH oxidase purified from the extreme thermophile Thermus thermoph ilus HB8 is a monomeric flavoprotein with a 1 : 1 ratio of flavin-aden ine dinucleotide (FAD) to the polypeptide chain. It catalyzes in vitro the oxidation of reduced NADH or NADPH with the formation of H2O2. Th e gene encoding the NADH oxidase from T. thermophilus HB8 was cloned, and its nucleotide sequence was determined. The molecular mass of 22,7 49 Da, as deduced from the nox gene, agrees with that of the purified NADH oxidase from T. thermophilus HB8, as estimated by mass spectromet ry. The nor gene does not contain a GX(4)GK consensus sequence typical for nucleotide binding proteins. The nox gene was overexpressed in Es cherichia coli, and a protocol for the rapid purification of the E. co li-borne T. thermophilus NADH oxidase or its His(6)-tagged analogue wa s developed by using thermal denaturation step and affinity chromatogr aphy.