Phosphorylated and dephosphorylated structures of pig heart, GTP-specific succinyl-CoA synthetase

In Archaea, acetate formation and ATP synthesis from acetylCoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA ؉ ADP ؉ P i % acetate ؉ ATP ؉ HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the ␤-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (␣ 2 ␤ 2 ) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257␣, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257␣ to ADP via transient phosphorylation of a second conserved histidine residue in the ␤-subunit, His-71␤. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues: ADP-forming acetyl-CoA synthetase (ACD) 2 (acetyl-CoA ϩ ADP ϩ P i % acetate ϩ ATP ϩ CoA) is a novel enzyme in Archaea that catalyzes the conversion of acetyl-CoA and other acyl-CoA esters to the corresponding acids and couples this reaction with the synthesis of ATP. This unusual synthetase has first been detected in the eukaryotic protists Entamoeba histolytica and Giardia lamblia (1, 2). In our group, ACD activities have been identified in all acetate-forming Archaea tested, including anaerobic hyperthermophiles and aerobic halophiles (3, 4). The conversion of acetyl-CoA to acetate by one enzyme is unusual in prokaryotes and appears to be restricted to Archaea, since all acetate-forming bacteria, including the hyperthermophile Thermotoga maritima, utilize two enzymes, phosphate acetyltransferase and acetate kinase, for acetate formation and ATP synthesis (4, 5). In anaerobic hyperthermophilic Archaea, e.g. Pyrococcus furiosus, ACD represents the major energy-conserving reaction during peptide, pyruvate, and sugar fermentation to acetate (4, 6, 7). Two ACD isoenzymes, ACD I and ACD II, have been characterized from P. furiosus, which differ in their substrate specificity for CoA esters. ACD I preferentially utilized acetyl-CoA and is involved in sugar degradation, whereas ACD II is involved in peptide fermentation (8).Subsequently, ACDs have been...

Section: Discussionmentioning

confidence: 99%

Reaction Mechanism and Structural Model of ADP-forming Acetyl-CoA Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Bräsen

Schmidt

Grötzinger

et al. 2008

“…8), and there are numerous examples of other structurally unrelated domains with histidine phosphotransfer functions that are not part of two-component signal transduction systems. These would include the phospho-histidine proteins that mediate sugar transport and carbohydrate repression in bacteria (42) as well as a large number of metabolic enzymes such as nucleoside diphosphate kinase (43) and succinyl-CoA synthetase (44). Moreover, the CheA dimerization domain, which does not have a histidine phosphotransfer activity, seems homologous to the H box-containing dimerization domains of histidine kinases such as EnvZ (4,8).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of the CheA Histidine Phosphotransfer Domain that Mediates Response Regulator Phosphorylation in Bacterial Chemotaxis

Mourey¹,

Re²,

Pédelacq³

et al. 2001

The x-ray crystal structure of the P1 or H domain of the Salmonella CheA protein has been solved at 2.1-Å resolution. The structure is composed of an up-down up-down four-helix bundle that is typical of histidine phosphotransfer or HPt domains such as Escherichia coli ArcB C and Saccharomyces cerevisiae Ypd1. Loop regions and additional structural features distinguish all three proteins. The CheA domain has an additional Cterminal helix that lies over the surface formed by the C and D helices. The phosphoaccepting His-48 is located at a solvent-exposed position in the middle of the B helix where it is surrounded by several residues that are characteristic of other HPt domains. Mutagenesis studies indicate that conserved glutamate and lysine residues that are part of a hydrogen-bond network with His-48 are essential for the ATP-dependent phosphorylation reaction but not for the phosphotransfer reaction with CheY. These results suggest that the CheA-P1 domain may serve as a good model for understanding the general function of HPt domains in complex two-component phosphorelay systems.The signal transduction system that controls motor behavior in Salmonella and Escherichia coli provides a paradigm for understanding the biochemistry of intracellular information processing networks (for recent reviews see Refs.

“…The ␤-subunit begins with the amino acids MNLQ and the aminoterminal methionine residue is not cleaved. The design of the gene is different from one used earlier (37) because the crystal structure showed that a methionine residue inserted before the first residue of the mature ␤-subunit could interfere with catalysis (36). It may be the reason for the reduced specific activity of the enzyme produced in E. coli (37).…”

Section: Methodsmentioning

confidence: 97%

“…The structures were solved by molecular replacement using the model of pig GTP-specific SCS identified in the Protein Data Bank (43) as 1EUD (36) and the program AMoRe (44) or by difference Fourier techniques. There is one molecule per asymmetric unit.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Interactions of GTP with the ATP-grasp Domain of GTP-specific Succinyl-CoA Synthetase

Fraser

Hayakawa

Hume

et al. 2006

Self Cite

Two isoforms of succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 Å resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20␤) and with backbone atoms provide the specificity. The ␥-phosphate interacts with the side chain of an arginine residue (Arg-54␤) and with backbone amide nitrogen atoms, leading to tight interactions between the ␥-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the ␥-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the ␣-and ␤-phosphates allow the ␥-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the ␤-phosphate interacts with these residues in GDP. The complex of GTP with succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.The enzyme succinyl-CoA synthetase (SCS) 4 uses ATP or GTP to catalyze the formation of succinyl-CoA from succinate and coenzyme A. In animals, two different isoforms exist, one specific for ATP and the other specific for GTP (1). The two isoforms include the same ␣-subunit, but different ␤-subunits (2). The amino-terminal domain of the ␤-subunit has an ATP-grasp fold (3-5), and Mg 2ϩ -ADP was shown to bind in this domain of the Escherichia coli SCS using labeling experiments and site-directed mutagenesis (6) and by soaking the nucleotide into crystals and determining the structure of the resulting complex (7). The ATP-grasp fold has been found in a number of other enzymes (8) (24), and RNA ligase 2 (25). The determinations of the structures of several of these enzymes in complex with nucleotides, nucleotide analogues, and their other substrates has led to a good understanding of the interactions that are important in their binding and catalysis.The biological roles of the ATP-and GTP-specific SCS have not been fully delineated. Originally it was thought that the primary role for SCS was in the citric acid cycle, where it was responsible for the breakdown of succinyl-CoA to succinate and coenzyme A accompanied by the phosphorylation of nucleotide diphosphate to nucleotide triphosphate (26). This step provides the only substrate-level phosphorylation of the citric acid cycle. It was thought that some species had SCS that could use either ADP or GDP, e.g. E. coli (27), while others had SCS that coul...