Cloning and Characterization of Two NAD Kinases from Arabidopsis. Identification of a Calmodulin Binding Isoform

NAD kinase (NADK; ATP:NAD 2ʹ-phosphotransferase, EC 2.7.1.23), an enzyme found in both prokaryotes and eukaryotes, generates the important pyridine nucleotide NADP from substrates ATP and NAD. The role of NADKs in plants is poorly understood, and cDNAs encoding plant NADKs have not previously been described to our knowledge. We have cloned two cDNAs from Arabidopsis predicted to encode NADK isoforms, designated NADK1 and NADK2, respectively. Expressed as recombinant proteins in bacteria, both NADK1 and NADK2 were catalytically active, thereby confirming their identity as NADKs. Transcripts for both isoforms were detected in all tissues examined and throughout development. Although the predicted catalytic regions for NADK1 and NADK2 show sequence similarity to NADKs from other organisms, NADK2 possesses a large N-terminal extension that appears to be unique to plants. Using recombinant glutathione-S-transferase fusion proteins and calmodulin (CaM)affinity chromatography, we delineated a Ca^2+-dependent CaM-binding domain to a 45-residue region within the Nterminal extension of NADK2. Although recombinant NADK2 was not responsive to CaM in vitro, immunoblot analysis suggests that native NADK2 is a CaM-binding protein. In Arabidopsis crude extracts, CaM-dependent NADK activity was much greater than CaM-independent activity throughout development, particularly in young seedlings. A native CaM-dependent NADK was partially purified from Arabidopsis seedlings (KmNAD=0.20 mM, KmMg2+–ATP=0.17 mM). The enzyme was fully activated by conserved CaM (S0.5 = 2.2 nM) in the presence of calcium but displayed differential responsiveness to eight CaM-like Arabidopsis proteins. Possible roles for NADKs in plants are discussed in light of our observations.