Molecular Cloning and Characterization of a Candidate Plant Growth-Related and Time-Keeping Constitutive Cell Surface Hydroquinone (NADH) Oxidase (ENOX1) from iArabidopsis lyrata/i

摘要

ENOX (ECTO-NOX) proteins are proteins of the external surface of the plasma membrane that catalyze oxidation of both NADH and hydroquinones as well as carry out protein disulfidethiol interchange. They exhibit both prion-like and time-keeping (clock) properties. The oxidative and interchange activities alternate to generate a regular period of 24 min in length. Here we report the cloning, expression, and characterization of a plant candidate constitutive ENOX (CNOX or ENOX1) protein from Arabidopsis lyrata. The gene encoding the 335 (165) amino acid protein is found in accession XP-002882467. Functional motifs characteristics of ENOX proteins previously identified by site-directed mutagenesis and present in the candidate ENOX1 protein from plants include adenine nucleotide and copper binding motifs along with essential cysteines. However, the drug binding motif (EEMTE) sequence of human ENOX2 is absent. The activities of the recombinant protein expressed in E. coli were unaffected by capsaicin, EGCg, and other ENOX2-inhibiting substances. Periodic oxidative activity was exhibited both with NAD(P)H and reduced coenzyme Q as substrate. Bound copper was necessary for activity and activity was inhibited by the ENOX1-specific inhibitor simalikalactone D. Addition of melatonin phased the 24-min period such that the next complete period began 24 min after the melatonin addition as appeared to be characteristic of ENOX1 activities in general. Periodic protein disulfide-thiol interchange activity also was demonstrated along with the 2 oxidative plus 3 interchange activity pattern characteristics of the 24-min ENOX1 protein period. Concentrated solutions of the purified plant ENOX1 protein formed insoluble aggregates, devoid of enzymatic activity, resembling amyloid. Activity was restored to aggregate preparations by isoelectric focusing.