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

Abstract

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 disulfide-thiol 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 * Corresponding author. X. Y. Tang et al.2 aggregate preparations by isoelectric focusing.

Cloning and Characterization of a Candidate Auxin Plant Growth Regulator-Activated Cell Surface Hydroquinone (NADH) Oxidase

“…The sequence of the putative plant auxin-activated ENOX protein appeared not to resemble that of ENOX proteins previously described. Except for putative functional motifs, no similarity to the human ENOX2 [6] or ENOX1 [4], the plant ENOX1 [22] or the yeast ENOX1 [21] was found. A putative NADH-binding site, a disulfide-thiol interchange site, and copper-binding sites of ENOX2 are conserved in the human ENOX1 and ENOX2 proteins while the putative anti-cancer drug binding site of ENOX2 is not.…”

“…Polyclonal antibodies raised to the fragment blocked auxin-responsive NADH oxidase and auxin binding of plant plasma membrane vesicles but failed to yield immunoreactive clones upon expression cloning. Subsequent cloning of ENOX1 proteins first from yeast [21] and then from Arabidopsis [22] provided sequence information to identify potential functional motifs. These motifs, when located in a single polypeptide chain together with that of a known auxin-binding motif, resulted in the identification of a candidate 213 amino acid sequence ABP-20 from Prunus persicaria [23] that contained an auxin binding motif with homology to the auxin binding site of ABP1 [24] but of different specificity (2,4-D > PCIB > NAA = IAA).…”