P36, a Dictyostelium discoideum protein whose phosphorylation is stimulated by GDP, is homologous to the α-subunit of succinyl-CoA synthetase

Anschutz, Alison; Um, Hong‐Duck; Siegel, Ned R.; Véron, Michel; Klein, Claudette

doi:10.1016/0167-4838(93)90125-b

Cited by 14 publications

(13 citation statements)

References 23 publications

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“…As shown in Fig. 4B, the N-terminal sequence of the isolated 33-kDa protein subunit was found to be identical at 11 out of 12 residues to the a subunit of Scs isolated from D. discoideum (18) and at 9 out of 12 residues to the a subunit of Scs isolated from E. The N-terminal sequence of the isolated 33-kDa protein is identical at 11 out of 12 and 9 out of 12 residues to the a subunit of Scs proteins isolated from D. discoideum (18) and E. coli (19), respectively. The N-terminal sequence of the isolated 43-kDa protein is identical at 13 out of 15 residues to the (3 subunit of the Scs protein isolated from E. coli (19).…”

Section: Methodsmentioning

confidence: 90%

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Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa: complex formation with succinyl-CoA synthetase.

Kavanaugh-Black

Connolly

Chugani

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The enzyme nucleoside-diphosphate kinase (Ndk), responsible for the conversion of (deoxy)ribonucleoside diphosphates to their corresponding triphosphates, has been purified from Pseudomonas aeruginosa. The N-terminal 12 amino acid sequence of P. aeruginosa Ndk shows s nt homology with that of Myxococcus xanthus and that of Eschernchia colt. Ndk enzyme activity is also associated with succinyl-CoA synthetase activity in P. aeruginosa, whose a and 13 subunits show extensive sequence homology with those of E.coli and Dictyosteium discoideum. 7). Expression of nm23 from a constitutive promoter in highly metastatic murine tumor cells was found to actually suppress tumor metastasis (8). Null mutations in the Ndk gene of Drosophila, named awd, cause abnormalities in development of the larvae leading to tissue necrosis and death at the prepupal stage (7, 9). In the slime mold Dictyostelium discoideum, the Ndk gene is developmentally regulated with a sharp decrease in Ndk transcript levels coinciding with the onset ofthe starvation induced developmental cycle (10, 11). A gene encoding a DNA-binding protein, PuF, which is required for transcription of c-myc in vitro, is highly homologous to the human Ndk gene nm23-H2 (12). This implies that nm23-H2 may be involved in the regulation of c-myc.Pseudomonas aeruginosa is a ubiquitous soil and waterborne bacterium which undergoes a transition to mucoidy under stress and/or starvation conditions (13,14). We have purified Ndk from P. aeruginosa in order to study its involvement in these developmental changes and have found that Ndk is associated and copurifies with the ATP-utilizing prokaryotic succinyl-CoA synthetase (Scs) complex. These results indicate that aside from its role in DNA and RNA precursor synthesis, Ndk may be involved in regulating intracellular energy metabolism and that understanding its role in energy metabolism may be critical to understanding its involvement in regulation of development. MATERIALS AND METHODS Detection of [y-32P]ATP-Autophosphorylated Proteins.Samples were added to TMD buffer (50 mM Tris'HCl, pH 7.5/1 mM MgCl2/0.2 mM dithiothreitol) in <19 t4 and brought to a final volume of 20 id with TMD buffer.[y-32P]ATP (1G4; 74 MBq/ml) was added to the sample (final ATP concentration, 20 nM) and reactions were allowed to proceed for at least 6 sec. Reactions were terminated by addition of 4x SDS gel running buffer [0.2 M Tris HCl, pH 6.8/0.62% dithiothreitol/8% SDS/0.01% bromophenol blue/ 40o (vol/vol) glycerol] and analyzed by electrophoresis in an SDS/polyacrylamide gel (3% stacking gel, 15% separating gel).Purification of Ndk from P. aeruginosa strain 8822. Strain 8822 was grown overnight in Luria broth containing 0.1% glucose, and a crude cell extract was prepared from 4 liters of culture by French press disruption and ultracentrifugation at 300,000 x g for 1 hr. The supernatant was subjected to (NH4)2S04 fractionation at 45% and 65% saturation (2), and precipitates at 65% were suspended in 9.6 ml of TMD buffer containing 1 M (NH4)2SO4 (pH ...

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Section: Methodsmentioning

confidence: 90%

“…Succinyl-CoA synthetase has been variously described as utilizing ATP alone as a substrate in catalysis (21) or as utilizing ATP, GTP, and ITP, in that order, as substrates in its reaction (22). These (18). These starvation conditions are also cited as inducing a sharp decrease in ndk transcript levels in D. discoideum (10,11).…”

mentioning

confidence: 99%

Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa: complex formation with succinyl-CoA synthetase.

Kavanaugh-Black

Connolly

Chugani

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…In contrast, Scs from eukaryotes such as pig heart (8) or Dictyostelium discoidium (1,25,26) is known to generate GTP. However, both ATP-and GTP-specific Scs isoforms have been characterized from multicellular eukaryotes (8) and, in general, the nucleotide preference varies widely within organisms (11,20).…”

Section: Discussionmentioning

confidence: 98%

Succinyl Coenzyme A Synthetase of Pseudomonas aeruginosa with a Broad Specificity for Nucleoside Triphosphate (NTP) Synthesis Modulates Specificity for NTP Synthesis by the 12-Kilodalton Form of Nucleoside Diphosphate Kinase

2000

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Pseudomonas aeruginosa secretes copious amounts of an exopolysaccharide called alginate during infection in the lungs of cystic fibrosis patients. A mutation in the algR2 gene of mucoid P. aeruginosa is known to exhibit a nonmucoid (nonalginate-producing) phenotype and showed reduced activities of succinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate kinase (Ndk), implying coregulation of Ndk and Scs in alginate synthesis. We have cloned and characterized the sucCD operon encoding the ␣ and ␤ subunits of Scs from P. aeruginosa and have studied the role of Scs in generating GTP, an important precursor in alginate synthesis. We demonstrate that, in the presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor and, in the presence of ADP, Scs synthesizes ATP using GTP as a phosphodonor. In the presence of inorganic orthophosphate, succinyl-CoA, and an equimolar amount of ADP and GDP, Scs synthesizes essentially an equimolar amount of ATP and GTP. Such a mechanism of GTP synthesis can be an alternate source for the synthesis of alginate as well as for the synthesis of other macromolecules requiring GTP such as RNA and protein. Scs from P. aeruginosa is also shown to exhibit a broad NDP kinase activity. In the presence of inorganic orthophosphate (P i ), succinyl-CoA, and either GDP, ADP, UDP or CDP, it synthesizes GTP, ATP, UTP, or CTP. Scs was previously shown to copurify with Ndk, presumably as a complex. In mucoid cells of P. aeruginosa, Ndk is also known to exist in two forms, a 16-kDa cytoplasmic form predominant in the log phase and a 12-kDa membrane-associated form predominant in the stationary phase. We have observed that the 16-kDa Ndk-Scs complex present in nonmucoid cells, synthesizes all three of the nucleoside triphosphates from a mixture of GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifically present in mucoid cell predominantly synthesizes GTP and UTP but not CTP. Such regulation may promote GTP synthesis in the stationary phase when the bulk of alginate is synthesized by mucoid P. aeruginosa.Our laboratory is interested in studying the regulation of biosynthesis of alginate in Pseudomonas aeruginosa, an opportunistic pathogen. A number of cystic fibrosis (CF) isolates have been studied for their enhanced synthesis and secretion of alginate (16). Alginate is a polymer of D-manuronic and its C-5 epimer L-guluronic acid. GDP-mannuronic acid, derived from GDP-mannose, is the building block of alginate, which requires mannose 1-phosphate and GTP as precursors (17). Thus, alginate synthesis demands a steady supply of GTP for every molecule of mannuronic acid incorporated into a molecule of alginate. Recently, Kamath et al. (9) have shown that the mucoid strains of P. aeruginosa retain an active form of elastase in their periplasm, and this periplasmic elastase acts on 16-kDa Ndk to generate a truncated 12-kDa form that is membrane associated. It has been demonstrated that the membrane-associated 12-kDa nucleoside diphosphate (NDP) kinase (Ndk), in complex with pyruvat...

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“…Protein determinations were done according to the method of Peterson (16). Partial purification of SCS on hydroxyapatite was performed as previously described (2). The stability of the radiolabel in the reaction mixture was monitored by thin-layer chromatography on polyethyleneimine-cellulose MN 300 plates in 0.75 M KH 2 PO 4 , pH 3.4, as previously described (19).…”

Section: Methodsmentioning

confidence: 99%

Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation

Birney

Klein

1996

J Bacteriol

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Low concentrations of ADP are shown to increase the rate of phosphoenzyme formation of E. coli succinylcoenzyme A (CoA) synthetase (SCS) without altering the fraction of phosphorylated enzyme. This is true when either ATP or succinyl-CoA and P i are used to phosphorylate the enzyme. The stimulatory effect of ADP is not altered by sample dilution, is retained upon partial purification of the enzyme, and reflects the binding of ADP to a site other than the catalytic site. GDP also alters the phosphorylation of the E. coli SCS but does so primarily by enhancing the level of the phosphoenzyme and only when ATP is used as the phosphate donor. GDP appears to function by neutralizing the action of a specific inhibitory protein. This inhibitor of SCS allows for interconversion of succinate and succinyl-CoA in a manner dissociated from changes in ATP-ADP metabolism. These previously unidentified and varied mechanisms by which SCS is regulated focus attention on this enzyme as an important control point in determining the cell's potential to meet its metabolic demands.The processes of cell differentiation, growth, and aging reflect the selection of specific pathways to be activated or inactivated. Such metabolic regulation may occur at the level of expression of components in those pathways or by modulating their activities. We have been investigating how changes in energy metabolism reflect or determine the cell's genetic potential for growth and development. In particular, our studies have focused on the regulation of succinyl-coenzyme A (CoA) synthetase (SCS), the only enzyme in the citric acid cycle that catalyzes a substrate-level phosphorylation reaction. This reaction is completely reversible, allowing for the production of nucleoside triphosphate (NTP) during aerobic metabolism and the synthesis of succinyl-CoA for anabolic reactions. Partial reactionswhere NDP is nucleoside diphosphate and E is the enzyme. As seen in the schema above, the phosphoenzyme is an intermediate in the reaction and can be formed by autophosphorylation with either an NTP (top partial reaction) or succinyl-CoA and P i . The enzyme is composed of two distinct protein subunits, ␣ and ␤. There are two known forms of the enzyme. In gram-negative bacteria such as Escherichia coli, the enzyme is a tetramer of two ␣ and two ␤ subunits. It preferentially uses adenine nucleotides and will be referred to here as the A-form enzyme. G-form enzymes, which appear to use exclusively guanine nucleotides in the reaction, predominate in eukaryotes and gram-positive bacteria. They function as dimers composed of one ␣ and one ␤ subunit. The physiological significance of the differences in quaternary structure and nucleotide preferences with respect to enzyme action or organization in the citric acid cycle is unknown. In either case, it is a histidine residue in the ␣ subunit that is phosphorylated to form the phosphoenzyme intermediate. (for reviews, see references 5 and 14).In the presence of saturating or near-saturating concentrations of substrates, SCSs f...

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P36, a Dictyostelium discoideum protein whose phosphorylation is stimulated by GDP, is homologous to the α-subunit of succinyl-CoA synthetase

Cited by 14 publications

References 23 publications

Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa: complex formation with succinyl-CoA synthetase.

Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa: complex formation with succinyl-CoA synthetase.

Succinyl Coenzyme A Synthetase of Pseudomonas aeruginosa with a Broad Specificity for Nucleoside Triphosphate (NTP) Synthesis Modulates Specificity for NTP Synthesis by the 12-Kilodalton Form of Nucleoside Diphosphate Kinase

Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation

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