A new mitochondrial three-component pyridine nucleotide pathway, namely a NMN→NAD→NADP route, is functional in plant heterotrophic tissues

The multiple roles of NAD(P) in bioenergetics and production of secondary messengers as well as in protein modifications and generation of free and protein-associated poly-ADP-ribose require a constant resynthesis of NAD(P) to avoid depletion of the intracellular NAD(P) pool. Nicotinamide mononucleotide adenylyl-transferase (NMN-AT) (EC 2.7.7.1) is a central enzyme in NAD+ biosynthesis. It catalyzes the reversible reaction NMN+ATP↔NAD++PPi NADP+ is produced by NAD+ kinase (EC 2.7.1.23): NAD++ATP↔NADP++ADP. The aim of this study was to investigate whether mitochondria isolated from plant heterotrophic tissues can synthesize nicotinamide adenine dinucleotides from externally added NMN. The experimental work was articulated in the following phases: First, plant mitochondria were checked with respect to their functional features (intactness of mitochondrial outer membrane, intactness of mitochondrial inner membrane, Respiratory Control, ΔΨm generation). Next, total NAD(P) was extracted from freshly isolated mitochondria and quantified by HPLC analysis. Plant mitochondria were incubated with NMN and ATP and the amount of NAD(P) synthesized was determined. Our findings show mitochondrial permeability to externally added NMN and a significant increase of newly synthesized NAD(P) in plant heterotrophic tissues . Moreover, we determined the kinetic parameters of the NMN-AT in fresh slices of plant heterotrophic tissues. Dependence of enzyme activity on pH and temperature and sensitivity to different inhibitors were also investigated. Based on our biochemical results, we discuss the profiles and the function of pyridine biosynthesis in plant mitochondria. PDF created