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

摘要

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 Mg²⁺ and NAD⁺, with respective Km values of 0.042, 0.062, 1.16, and 2.39 mM. While NADHK could phosphorylate NADH or NAD⁺, the specificity constant (Vmax/Km) 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. PPi or poly-Pi could not substitute as phospho donors. PPi 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.