Plants utilize a highly conserved system for repair of NADH and NADPH hydrates

NADH and NADPH undergo spontaneous and enzymatic reactions that produce R and S forms of NAD(P)H hydrates [NAD(P)HX], which are not electron donors and inhibit various dehydrogenases. In bacteria, yeast ( Saccharomyces cerevisiae ), and mammals, these hydrates are repaired by the tandem action of an ADP- or ATP-dependent dehydratase that converts ( S )-NAD(P)HX to NAD(P)H and an epimerase that facilitates interconversion of the R and S forms. Plants have homologs of both enzymes, the epimerase homolog being fused to the vitamin B 6 salvage enzyme pyridoxine 5′-phosphate oxidase. Recombinant maize ( Zea mays ) and Arabidopsis ( Arabidopsis thaliana ) NAD(P)HX dehydratases (GRMZM5G840928, At5g19150) were able to reconvert ( S )-NAD(P)HX to NAD(P)H in an ATP-dependent manner. Recombinant maize and Arabidopsis epimerases (GRMZM2G061988, At5g49970) rapidly interconverted ( R )- and ( S )-NAD(P)HX, as did a truncated form of the Arabidopsis epimerase lacking the pyridoxine 5′-phosphate oxidase domain. All plant NAD(P)HX dehydratase and epimerase sequences examined had predicted organellar targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays confirmed that both start sites were used. Dual import assays with purified pea ( Pisum sativum ) chloroplasts and mitochondria, and subcellular localization of GFP fusion constructs in tobacco ( Nicotiana tabacum ) suspension cells, indicated mitochondrial, plastidial, and cytosolic localization of the Arabidopsis epimerase and dehydratase. Ablation of the Arabidopsis dehydratase gene raised seedling levels of all NADHX forms by 20- to 40-fold, and levels of one NADPHX form by 10- to 30-fold. We conclude that plants have a canonical two-enzyme NAD(P)HX repair system that is directed to three subcellular compartments via the use of alternative translation start sites.