The structure of succinyl-CoA synthetase bound to the succinyl-phosphate intermediate clarifies the catalytic mechanism of ATP-citrate lyase.

Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85 Šresolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105αQ, was made in human GTP-specific SCS. The structure of the E105αQ mutant shows succinyl-phosphate bound to the enzyme at 1.58 Šresolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105αQ mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold and 11-fold higher, respectively. Based on this high-resolution structure, the cryo-EM maps of the E599Q ACLY complex reported previously should have revealed the binding of citryl-phosphate and CoA and not phospho-citryl-CoA.

Novel compound heterozygous SUCLG1 variants may contribute to mitochondria DNA depletion syndrome-9.

BACKGROUND: Succinate-CoA ligase/synthetase (SCS) deficiency is responsible for encephalomyopathy with mitochondrial DNA depletion and mild methylmalonic aciduria. Variants in SUCLG1, the nuclear gene encoding the alpha subunit of the SCS enzyme playing a pivotal role in maintaining mtDNA integrity and stability, are associated with mitochondrial DNA depletion syndrome 9 (MTDPS9). METHODS: In this study, we reported an infant with clinical features of MTDPS9 from China. Whole exome sequencing (WES) was used to identify the genetic cause. Bioinformatic analysis and mtDNA level detection were performed to assess pathogenicity. RESULTS: The proband manifested with hypotonia, lactic acidosis, mild methylmalonic aciduria, hearing loss and psychomotor retardation. WES identified new compound heterozygous SUCLG1 variants of c.601A>G (p.R201G) in exon 6 and c.871G>C (p.A291P) in exon 8. Computational analysis predicted that these missense variants might alter structure stability and mitochondrial translocation of SUCLG1. qRT-PCR showed 68% depletion of mtDNA content in proband as compared to controls. CONCLUSION: Novel compound heterozygous variants c.601A>G (p.R201G) and c.871G>C (p.A291P) in SUCLG1 may cause MTDPS9 in this family. Our finding should be helpful for molecular diagnosis, genetic counseling and clinical management of SCS deficiency disorders.

Second distinct conformation of the phosphohistidine loop in succinyl-CoA synthetase.

Succinyl-CoA synthetase (SCS) catalyzes a reversible reaction that is the only substrate-level phosphorylation in the citric acid cycle. One of the essential steps for the transfer of the phosphoryl group involves the movement of the phosphohistidine loop between active site I, where CoA, succinate and phosphate bind, and active site II, where the nucleotide binds. Here, the first crystal structure of SCS revealing the conformation of the phosphohistidine loop in site II of the porcine GTP-specific enzyme is presented. The phosphoryl transfer bridges a distance of 29 Šbetween the binding sites for phosphohistidine in site I and site II, so these crystal structures support the proposed mechanism of catalysis by SCS. In addition, a second succinate-binding site was discovered at the interface between the α- and ß-subunits of SCS, and another magnesium ion was found that interacts with the side chains of Glu141ß and Glu204ß via water-mediated interactions. These glutamate residues interact with the active-site histidine residue when it is bound in site II.

The nucleotide specificity of succinyl-CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone.

Substrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl-CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been shown to alter the substrate specificity via electrostatic interactions with approaching substrates. The enzyme kinetics of recombinant PfSCS (wild-type), generated by refolding of the individual P. falciparum SCSß and Blastocystis SCSα subunits, demonstrated ADP-forming activity (KmATP  = 48 µm). Further, the introduction of charged gatekeeper residues, either positive (Lys and Lys) or negative (Glu and Asp), resulted in significant reductions in the ATP affinity of PfSCS. It is interesting to note that the recombinant PfSCSß subunit can be refolded to a functional enzyme conformation using Blastocystis SCSα, indicating the possibility of subunits swapping among different organisms. These results concluded that electrostatic interactions at the gatekeeper region alone are insufficient to alter the substrate specificity of PfSCS, and further structural analysis with a particular focus on binding site architecture is required.

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei.

Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro-reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher kcat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.

Tartryl-CoA inhibits succinyl-CoA synthetase.

Succinyl-CoA synthetase (SCS) catalyzes the only substrate-level phosphorylation step in the tricarboxylic acid cycle. Human GTP-specific SCS (GTPSCS), an αß-heterodimer, was produced in Escherichia coli. The purified protein crystallized from a solution containing tartrate, CoA and magnesium chloride, and a crystal diffracted to 1.52 Šresolution. Tartryl-CoA was discovered to be bound to GTPSCS. The CoA portion lies in the amino-terminal domain of the α-subunit and the tartryl end extends towards the catalytic histidine residue. The terminal carboxylate binds to the phosphate-binding site of GTPSCS.

ATP-specificity of succinyl-CoA synthetase from Blastocystis hominis.

Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46ßE, K114ßD, V113ßL and L227ßF, and one with two mutations, K46ßE/K114ßD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20ß, Gly111ß and Val113ß. The O atom of the side chain of Gln20ß interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111ß. It is the different conformations of the backbone at Gln20ß, of the side chain of Gln20ß and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227ßF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46ßE/K114ßD double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.

Structure-Based Design, Synthesis, and Biological Evaluation of Non-Acyl Sulfamate Inhibitors of the Adenylate-Forming Enzyme MenE.

N-Acyl sulfamoyladenosines (acyl-AMS) have been used extensively to inhibit adenylate-forming enzymes that are involved in a wide range of biological processes. These acyl-AMS inhibitors are nonhydrolyzable mimics of the cognate acyl adenylate intermediates that are bound tightly by adenylate-forming enzymes. However, the anionic acyl sulfamate moiety presents a pharmacological liability that may be detrimental to cell permeability and pharmacokinetic profiles. We have previously developed the acyl sulfamate OSB-AMS (1) as a potent inhibitor of the adenylate-forming enzyme MenE, an o-succinylbenzoate-CoA (OSB-CoA) synthetase that is required for bacterial menaquinone biosynthesis. Herein, we report the use of computational docking to develop novel, non-acyl sulfamate inhibitors of MenE. A m-phenyl ether-linked analogue (5) was found to be the most potent inhibitor (IC50 = 8 µM; Kd = 244 nM), and its X-ray co-crystal structure was determined to characterize its binding mode in comparison to the computational prediction. This work provides a framework for the development of potent non-acyl sulfamate inhibitors of other adenylate-forming enzymes in the future.

Clinical, Molecular, and Computational Analysis in two cases with mitochondrial encephalomyopathy associated with SUCLG1 mutation in a consanguineous family.

Deficiency of the mitochondrial enzyme succinyl COA ligase (SUCL) is associated with encephalomyopathic mtDNA depletion syndrome and methylmalonic aciduria. This disorder is caused by mutations in both SUCL subunits genes: SUCLG1 (α subnit) and SUCLA2 (ß subnit). We report here, two Tunisian patients belonging to a consanguineous family with mitochondrial encephalomyopathy, hearing loss, lactic acidosis, hypotonia, psychomotor retardation and methylmalonic aciduria. Mutational analysis of SUCLG1 gene showed, for the first time, the presence of c.41T > C in the exon 1 at homozygous state. In-silico analysis revealed that this mutation substitutes a conserved methionine residue to a threonine at position 14 (p.M14T) located at the SUCLG1 protein mitochondrial targeting sequence. Moreover, these analysis predicted that this mutation alter stability structure and mitochondrial translocation of the protein. In Addition, a decrease in mtDNA copy number was revealed by real time PCR in the peripheral blood leukocytes in the two patients compared with controls.

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode.

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Mechanistic Insights from the Crystal Structure of Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase Complexed with the Adenylate Intermediate.

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase, or MenE, catalyzes an essential step in vitamin K biosynthesis and is a valuable drug target. Like many other adenylating enzymes, it changes its structure to accommodate substrate binding, catalysis, and product release along the path of a domain alternation catalytic mechanism. We have determined the crystal structure of its complex with the adenylation product, o-succinylbenzoyl-adenosine monophosphate (OSB-AMP), and captured a new postadenylation state. This structure presents unique features such as a strained conformation for the bound adenylate intermediate to indicate that it represents the enzyme state after completion of the adenylation reaction but before release of the C domain in its transition to the thioesterification conformation. By comparison to the ATP-bound preadenylation conformation, structural changes are identified in both the reactants and the active site to allow inference about how these changes accommodate and facilitate the adenylation reaction and to directly support an in-line backside attack nucleophilic substitution mechanism for the first half-reaction. Mutational analysis suggests that the conserved His196 plays an important role in desolvation of the active site rather than stabilizing the transition state of the adenylation reaction. In addition, comparison of the new structure with a previously determined OSB-AMP-bound structure of the same enzyme allows us to propose a release mechanism of the C domain in its alteration to form the thioesterification conformation. These findings allow us to better understand the domain alternation catalytic mechanism of MenE as well as many other adenylating enzymes.

Structural basis for the binding of succinate to succinyl-CoA synthetase.

Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Šresolution. Succinate binds in the carboxy-terminal domain of the ß-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate.

Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents.

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

Structure of GTP-specific succinyl-CoA synthetase in complex with CoA.

Pig GTP-specific succinyl-CoA synthetase is an αß-heterodimer. The crystal structure of the complex with the substrate CoA was determined at 2.1 Šresolution. The structure shows CoA bound to the amino-terminal domain of the α-subunit, with the free thiol extending from the adenine portion into the site where the catalytic histidine residue resides.

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase.

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes.

Mitochondrial encephalomyopathy and retinoblastoma explained by compound heterozygosity of SUCLA2 point mutation and 13q14 deletion.

Mutations in SUCLA2, encoding the ß-subunit of succinyl-CoA synthetase of Krebs cycle, are one cause of mitochondrial DNA depletion syndrome. Patients have been reported to have severe progressive childhood-onset encephalomyopathy, and methylmalonic aciduria, often leading to death in childhood. We studied two families, with children manifesting with slowly progressive mitochondrial encephalomyopathy, hearing impairment and transient methylmalonic aciduria, without mtDNA depletion. The other family also showed dominant inheritance of bilateral retinoblastoma, which coexisted with mitochondrial encephalomyopathy in one patient. We found a variant in SUCLA2 leading to Asp333Gly change, homozygous in one patient and compound heterozygous in one. The latter patient also carried a deletion of 13q14 of the other allele, discovered with molecular karyotyping. The deletion spanned both SUCLA2 and RB1 gene regions, leading to manifestation of both mitochondrial disease and retinoblastoma. We made a homology model for human succinyl-CoA synthetase and used it for structure-function analysis of all reported pathogenic mutations in SUCLA2. On the basis of our model, all previously described mutations were predicted to result in decreased amounts of incorrectly assembled protein or disruption of ADP phosphorylation, explaining the severe early lethal manifestations. However, the Asp333Gly change was predicted to reduce the activity of the otherwise functional enzyme. On the basis of our findings, SUCLA2 mutations should be analyzed in patients with slowly progressive encephalomyopathy, even in the absence of methylmalonic aciduria or mitochondrial DNA depletion. In addition, an encephalomyopathy in a patient with retinoblastoma suggests mutations affecting SUCLA2.

The novel mutation p.Asp251Asn in the ß-subunit of succinate-CoA ligase causes encephalomyopathy and elevated succinylcarnitine.

SUCLA2 is one of several nuclear-encoded genes that can cause encephalomyopathy accompanied by mitochondrial DNA depletion. The disorder usually manifests in early childhood and leads to early death. The gene encodes one of the subunits of succinyl-CoA synthase, the enzyme that catalyzes the reversible conversion of substrates succinyl-CoA and ADP to products succinate and ATP in the tricarboxylic acid pathway. Thirty-two individuals harboring mutations in SUCLA2 have so far been reported, and five different mutations were observed among these individuals. Here we report identification of a novel mutation in SUCLA2 in two cousins affected with encephalomyopathy. The novel mutation causes p.Asp251Asn; the affected amino acid is likely positioned within the ATP-grasp domain of the encoded protein. As previously reported in other patients, we did not observe elevation of methylmalonic acid, the biochemical hallmark of patients with mutations in SUCLA2. We instead found elevated levels of succinylcarnitine.

Identification of the kinetic mechanism of succinyl-CoA synthetase.

The kinetic mechanism of SCS [succinyl-CoA (coenzyme A) synthetase], which participates in the TCA (tricarboxylic acid) cycle, ketone body metabolism and haem biosynthesis, has not been fully characterized. Namely, a representative catalytic mechanism and associated kinetic parameters that can explain data on the enzyme-catalysed reaction kinetics have not been established. To determine an accurate model, a set of putative mechanisms of SCS, proposed by previous researchers, were tested against experimental data (from previous publication) on SCS derived from porcine myocardium. Based on comparisons between model simulation and the experimental data, an ordered ter-ter mechanism with dead-end product inhibition of succinate against succinyl-CoA is determined to be the best candidate mechanism. A thermodynamically constrained set of parameter values is identified for this candidate mechanism.

Biochemical and structural characterization of the GTP-preferring succinyl-CoA synthetase from Thermus aquaticus.

Succinyl-CoA synthetase (SCS) from Thermus aquaticus was characterized biochemically via measurements of the activity of the enzyme and determination of its quaternary structure as well as its stability and refolding properties. The enzyme is most active between pH 8.0 and 8.4 and its activity increases with temperature to about 339 K. Gel-filtration chromatography and sedimentation equilibrium under native conditions demonstrated that the enzyme is a heterotetramer of two α-subunits and two ß-subunits. The activity assays showed that the enzyme uses either ADP/ATP or GDP/GTP, but prefers GDP/GTP. This contrasts with Escherichia coli SCS, which uses GDP/GTP but prefers ADP/ATP. To understand the nucleotide preference, T. aquaticus SCS was crystallized in the presence of GDP, leading to the determination of the structure in complex with GDP-Mn(2+). A water molecule and Pro20ß in T. aquaticus take the place of Gln20ß in pig GTP-specific SCS, interacting well with the guanine base and other residues of the nucleotide-binding site. This leads to the preference for GDP/GTP, but does not hinder the binding of ADP/ATP.