Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress)

Abstract

NADH kinase (NADHK; ATP:NADH 2 -phosphotransferase; EC 2.7.1.86), an enzyme that preferentially utilizes NADH as the diphosphonicotinamide nucleotide donor, has been identified for the first time in plants. Low activity (0.4 nmol of NADPH produced/min per mg of protein) was observed in clarified protein extracts from Arabidopsis thaliana (thale cress) cell suspension cultures. However, unlike an NADHK from yeast (Saccharomyces cerevisiae) (POS5), the enzyme from Arabidopsis did not associate with the mitochondria. NADHK was cloned (gi:30699338) from Arabidopsis and studied as a recombinant protein following affinity purification from Escherichia coli. The enzyme had a pH optimum for activity of 7.9 and a subunit molecular mass of 35 kDa. Analytical gel filtration demonstrated that the recombinant enzyme exists as a dimer. Hyperbolic saturation kinetics were observed for the binding of NADH, ATP, free Mg 2+ and NAD + , with respective K m values of 0.042, 0.062, 1.16, and 2.39 mM. While NADHK could phosphorylate NADH or NAD + , the specificity constant (V max /K m ) for NADH was 100-fold greater than for NAD + . The enzyme could utilize UTP, GTP and CTP as alternative nucleotides, although ATP was the preferred substrate. PP i or poly-P i could not substitute as phospho donors. PP i acted as a mixed inhibitor with respect to both NADH and ATP. NADHK was inactivated by thiol-modifying reagents, with inactivation being decreased in the presence of NADH or ATP, but not NAD + . This study suggests that, in Arabidopsis, NADP + /NADPH biosynthetic capacity could, under some circumstances, become uncoupled from the redox status of the diphosphonicotinamide nucleotide pool.

The power to reduce: pyridine nucleotides – small molecules with a multitude of functions

“…The subcellular location of NADK-3 is less clear. The protein has been suggested to be cytosolic, because immunoblot analyses did not reveal a corresponding reactive band in mitochondrial fractions [160]. Still, the NADK-3 sequence is similar to the yeast mitochondrial NADK-3 and also prefers NADH to NAD + as its substrate.…”

“…AtNADK-1 lacks any potential targeting sequence and is most probably cytosolic. AtNADK-2 and AtNADK-3 contain a putative chloroplast transit peptide and a mitochondrial targeting sequence respectively [159,160]. A green fluorescent protein-NADK-2 construct colocalized with chlorophyll [159].…”

Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas

“…A key factor, required to maintain this reduced environment, is NADPH. Major sources of NADPH supply in the cytosol of plant organisms are the glucose consuming oxidative pentose phosphate cycle (17) and NAD(P)H exporting shuttle systems in the chloroplast envelope membrane (17,20,21), which are reliant on photosynthetic activity in the plastid. In our current working model, physiological conditions characterized by a sufficient provision of these photosynthates are connected to an active state of NAB1 and hence effective translation repression of LHCII transcripts.…”

Structure and Function of NAD Kinase and NADP Phosphatase: Key Enzymes That Regulate the Intracellular Balance of NAD(H) and NADP(H)

“…Apart from the physiological aspects, structural details of the interaction are also unknown. However, recombinant A. thaliana NAD kinases (NADK1, NADK2, and NADK3) are Ca 2þ /calmodulin-independent 40,41) and not activated by Ca 2þ /calmodulin, although recombinant NADK2, which has a calmodulin-binding motif in its N-terminal, is able to bind to calmodulin. 41) In addition, Ca 2þ /calmodulin has no detectable effect on recombinant human NAD kinase.…”

“…PCC6301, and yeast S. cerevisiae have three homolog genes; and the yeast Candida albicans, the protist (a cellular slime mold) Dictyostelium discoideum, and some eubacteria (Bacillus, Listeria, Streptomyces, and other cyanobacteria) contain two homolog genes. We cloned NAD kinase homolog genes from several microorganisms, 21,22,26,[30][31][32][33][34] and other researchers have cloned them from microorganisms, 3,29,[35][36][37] plants, [38][39][40][41] and humans, 42) and the properties of the gene products (recombinant NAD kinases) were determined (summarized in Table 1). All characterized NAD kinases show homomultimer (homodimer, homotetramer, homohexamer, or homooctamer) structures.…”