Plants Utilize a Highly Conserved System for Repair of NADH and NADPH Hydrates    

Abstract

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 59-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 59-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.

A Pathway for Repair of NAD(P)H in Plants

“…The dehydratase and epimerase activities of NNRD and NNRE, respectively, were also performed and validated with NADPH, leading to very similar results (data not shown). These data are corroborated by an independent study that appeared while this manuscript was under review (24).…”

Balancing of B6 Vitamers Is Essential for Plant Development and Metabolism in Arabidopsis

“…However, no or extremely low levels of protein could be detected in either mutant line by immunochemical analysis using an antibody specifically raised against Arabidopsis PDX3 ( Figure 2C). By contrast, a strong protein band of ;55 kD could be detected in wild-type protein extracts consistent with the expected molecular mass of mature PDX3 (52.2 kD) (Colinas et al, 2014;Niehaus et al, 2014). Therefore, these two lines can be considered as strong pdx3 mutants.…”

“…In the salvage pathways of the plant model Arabidopsis thaliana, PN, PM, and PL can be phosphorylated by a kinase (named SALT OVERLY SENSITIVE4 [SOS4]; , and the phosphorylated forms, PNP and PMP, can be converted into PLP by the action of the oxidase PDX3 (González et al, 2007;Sang et al, 2007Sang et al, , 2011 (Figure 1). Interestingly, the PDX3 enzyme consists of two domains; the PMP/PNP oxidase domain is located at the C terminus and is fused to an N-terminal epimerase that has recently been implicated in nicotinamide nucleotide repair (annotated NNRE) (Marbaix et al, 2011;Colinas et al, 2014;Niehaus et al, 2014). In addition, a pyridoxal reductase converting PL into PN, found originally in yeast (Morita et al, 2004), has more recently been characterized in Arabidopsis (Herrero et al, 2011).…”

“…Biosynthesis of PLP de novo takes place in the cytosol, whereas the PLP salvage enzyme SOS4 has been reported to be plastidic (Rueschhoff et al, 2013). An early study reported a plastidic location for PDX3 (Sang et al, 2011), but it was more recently demonstrated to localize to mitochondria (Colinas et al, 2014;Niehaus et al, 2014). Taken together, the contribution of PDX3 in provisioning PLP within the subcellular pool and its role in growth and development remain to be elucidated.…”

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase

“…However, a proteomic study (Ito et al, 2011) found the At5g32470 protein in a cytosolic fraction. Prompted by this latter finding, we discovered that At5g32470 and all of the orthologous sequences examined include a methionine residue near the end of the targeting peptide (residue 47 in At5g32470) that could potentially serve as an alternative translation start site (Supplemental Figure 6), as reported for other plant proteins (Daras et al, 2014;Niehaus et al, 2014;Bohrer et al, 2015;Ellens et al, 2015). If used, this second translation start site would eliminate the putative mitochondrial targeting peptide.…”