Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (Helianthus tuberosus L.) heterotrophic tissues

Abstract

Current studies in plants suggest that the content of the coenzyme NAD is variable and potentially important in determining cell fate. In cases that implicate NAD consumption, re-synthesis must occur to maintain dinucleotide pools. Despite information on the pathways involved in NAD synthesis in plants, the existence of a mitochondrial nicotinamide mononucleotide adenylyltransferase (NMNAT) activity which catalyses NAD synthesis from nicotinamide mononucleotide (NMN) and ATP has not been reported. To verify the latter assumed pathway, experiments with purified and bioenergetically active mitochondria prepared from tubers of Jerusalem artichoke (Helianthus tuberosus L.) were performed. To determine whether NAD biosynthesis might occur, NMN was added to Jerusalem artichoke mitochondria (JAM) and NAD biosynthesis was tested by means of HPLC and spectroscopically. Our results indicate that JAM contain a specific NMNAT inhibited by Na-pyrophosphate, AMP and ADP-ribose. The dependence of NAD synthesis rate on NMN concentration shows saturation kinetics with K (m) and V (max) values of 82 ± 1.05 μM and 4.20 ± 0.20 nmol min(-1) mg(-1) protein, respectively. The enzyme's pH and temperature dependence were also investigated. Fractionation studies revealed that mitochondrial NMNAT activity was present in the soluble matrix fraction. The NAD pool needed constant replenishment that might be modulated by environmental inputs. Thus, the mitochondrion in heterotrophic plant tissues ensures NAD biosynthesis by NMNAT activity and helps to orchestrate NAD metabolic network in implementing the survival strategy of cells.

Constitutive Nuclear Localization of an Alternatively Spliced Sirtuin-2 Isoform

“…Interestingly, yeast and Arabidopsis thaliana express mitochondrial NAD + transporters but lack an NMNAT3 homolog. Conversely, Helianthus tuberosus L (Jersualem artichoke) has a mitochondrial NMNAT [158], but a BLAST search failed to identify a putative gene coding for a NAD + transporter of the Arabidopsis thaliana type. This may suggest that mitochondrial NAD + import and biosynthesis are two evolutionary separate mechanisms to establish the mitochondrial NAD + pool, but may be mutually exclusive.…”

Mitochondrial involvement to methylglyoxal detoxification: d-Lactate/Malate antiporter in Saccharomyces cerevisiae

“…Compartmental organisation of eukaryotic cells requires mechanisms for precise distribution of metabolites to their target organelles (Palmieri et al 2000;Di Martino and Pallotta 2011).…”

Evidence for the presence of a FAD pyrophosphatase and a FMN phosphohydrolase in yeast mitochondria: a possible role in flavin homeostasis

“…Finally, in the light of cytosolic riboflavin, FMN and FAD concentrations, as calculated by measuring both spheroplast and mitochondrial content via HPLC (see Figure ), data presented in this report strongly suggest that mitochondria play a major role in regulating the flavin pool in yeast and in relation to flavin homeostasis. Furthermore, the pathways are also frequently distributed through multiple subcellular compartments and this introduces the challenge of how intermediates in these pathways may be shuttled between enzymes in the pathway, and whether this compartmentalization has any function in regulation of the pathways (Lunn, ; Di Martino and Pallotta, ).…”