Molecular cloning and characterization of a candidate human growth-related and time-keeping constitutive cell surface hydroquinone (NADH) oxidase

Abstract

ENOX (ECTO-NOX) proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide-thiol interchange and exhibit both prion-like and time-keeping (clock) properties. The two enzymatic activities they catalyze alternate to generate a regular period of 24 min in length. Here we report the cloning, expression, and characterization of a human candidate constitutive ENOX (CNOX or ENOX1) protein. The gene encoding this 643 amino acid long protein is located on chromosome 13 (13q 14.11). Functional motifs previously identified by site-directed mutagenesis in a cancer-associated ENOX (tNOX or ENOX2) as adenine nucleotide or copper binding along with essential cysteines are present, but the drug-binding motif (EEMTE) sequence of ENOX2 is absent. The activities of the recombinant protein expressed in Escherichia coli were not affected by capsaicin, EGCg, and other ENOX2-inhibiting substances. The purified recombinant protein bound ca. 2 mol of copper/mol of protein. Bound copper was necessary for activity. H260 and H579 were required for copper binding as confirmed by site-directed mutagenesis, loss of copper-binding capacity, and resultant loss of enzymatic activity. Addition of melatonin phased the 24 min period such that the next complete period began exactly 24 min after the melatonin addition as appears to be characteristic of ENOX1 activities in general. Oxidative activity was exhibited with both NAD(P)H and reduced coenzyme Q as substrate. Concentrated solutions of the purified candidate ENOX1 protein irreversibly formed insoluble aggregates, devoid of enzymatic activity, resembling amyloid.