Structure and Mutational Analysis of a Plant Mitochondrial Nucleoside Diphosphate Kinase. Identification of Residues Involved in Serine Phosphorylation and Oligomerization

Abstract

We report the first crystal structure of a plant (Pisum sativum L. cv Oregon sugarpod) mitochondrial nucleoside diphosphate kinase. Similar to other eukaryotic nucleoside diphosphate kinases, the plant enzyme is a hexamer; the six monomers in the asymmetric unit are arranged as trimers of dimers. Different functions of the kinase have been correlated with the oligomeric structure and the phosphorylation of Ser residues. We show that the occurrence of Ser autophosphorylation depends on enzymatic activity. The mutation of the strictly conserved Ser-119 to Ala reduced the Ser phosphorylation to about one-half of that observed in wild type with only a modest change of enzyme activity. We also show that mutating another strictly conserved Ser, Ser-69, to Ala reduces the enzyme activity to 6% and 14% of wild-type using dCDP and dTDP as acceptors, respectively. Changes in the oligomerization pattern of the S69A mutant were observed by cross-linking experiments. A reduction in trimer formation and a change in the dimer interaction could be detected with a concomitant increase of tetramers. We conclude that the S69 mutant is involved in the stabilization of the oligomeric state of this plant nucleoside diphosphate kinase.Nucleoside diphosphate kinases (NDPKs) are ubiquitous enzymes involved in equilibration of the cellular nucleoside triphosphate (NTP, dNTP) pools. They transfer phosphate groups from NTPs to nucleoside diphosphates in the presence of divalent cations, preferably Mg 21 . The reaction involves the formation of a covalent intermediate, whereby the enzyme is phosphorylated at the catalytic His residue. NDPKs have broad substrate specificity and can use both ribo-and deoxyribonucleotides of purines or pyrimidines (Parks et al., 1973). NDPK isoforms can be found within most cellular compartments in eukaryotes. There are eight isoforms in humans and four annotated isoforms in the Arabidopsis genome.Additional roles for NDPKs in processes other than basic metabolism have emerged. This was first observed when decreased expression levels of a nonmetastasis protein, Nm23-H1, correlated with reduced metastasis in certain cancers (Steeg et al., 1988). Nm23-H1 was subsequently revealed to be a NDPK. NDPKs are now thought to be involved in processes such as control of cell proliferation (Cipollini et al., 1997), regulation of transcription (Postel et al., 1993;Ji et al., 1995), and protein phosphotransferase activities in humans and fungi (Engel et al., 1995;Wagner and Vu, 2000;Ogura et al., 2001). Plant NDPKs are also involved in intracellular signaling processes, including phytochrome A response (Choi et al., 1999), UV-B light signaling (Zimmermann et al., 1999), and hormone response (Nato et al., 1997;Novikova et al., 1999). The plant mitochondrial isoform has been implicated in heat-stress response (Escobar Galvis et al., 2001) and cAMP signaling (Knorpp and Håkansson, 1998;Laukens et al., 2001).NDPKs share primary, secondary, and tertiary structural similarity but differ in their quaternary structure. Eukaryotic N...

ROS resistance in Pisum sativum cv. Alaska: the involvement of nucleoside diphosphate kinase in oxidative stress responses via the regulation of antioxidants

“…This gain in R3-1 NDPK2 phospho-transfer activity (to NDP and itself) could be via histidine, which might be stimulated by the altered amino acid (Fig. 2b), similar to the dependency of serine autophosphorylation via histidine (Johansson et al 2004). However, the mechanism is still undefined.…”

The pea mitochondrial nucleoside diphosphate kinase cleaves DNA and RNA

“…Proteins were expressed in E. coli as described in Johansson et al [19]. The His‐tag was subsequently cleaved off using TeV protease (Invitrogen), and eliminated by purification on a His‐trap column (Amersham Pharmacia Biotech).…”

Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato

“…NDPK activity and protein have also been identified in mitochondria (Escobar Galvis et al 1999) and shown to be localized in the mitochondrial intermembrane space (Sweetlove et al 2001). Mitochondrial NDPK oligomerization state has recently been characterized by mutational analysis and crystallography (Johansson et al 2004). This isoform is probably involved in energy metabolism and stress response since it has been shown to interact with the mitochondrial adenine nucleotide translocator (Knorpp et al 2003) and a heat-inducible 86-kDa protein (Escobar Galvis et al 2001).…”