Kinetic and biochemical characterization of Plasmodium falciparum GMP synthetase

Plasmodium falciparum, the causative agent of the fatal form of malaria, synthesizes GMP primarily from IMP and, hence, needs active GMPS (GMP synthetase) for its survival. GMPS, a G-type amidotransferase, catalyses the amination of XMP to GMP with the reaction occurring in two domains, the GAT (glutamine amidotransferase) and ATPPase (ATP pyrophosphatase). The GAT domain hydrolyses glutamine to glutamate and ammonia, while the ATPPase domain catalyses the formation of the intermediate AMP-XMP from ATP and XMP. Co-ordination of activity across the two domains, achieved through channelling of ammonia from GAT to the effector domain, is the hallmark of amidotransferases. Our studies aimed at understanding the kinetic mechanism of PfGMPS (Plasmodium falciparum GMPS) indicated steady-state ordered binding of ATP followed by XMP to the ATPPase domain with glutamine binding in a random manner to the GAT domain. We attribute the irreversible, Ping Pong step seen in initial velocity kinetics to the release of glutamate before the attack of the adenyl-XMP intermediate by ammonia. Specific aspects of the overall kinetic mechanism of PfGMPS are different from that reported for the human and Escherichia coli enzymes. Unlike human GMPS, absence of tight co-ordination of activity across the two domains was evident in the parasite enzyme. Variations seen in the inhibition by nucleosides and nucleotide analogues between human GMPS and PfGMPS highlighted differences in ligand specificity that could serve as a basis for the design of specific inhibitors. The present study represents the first report on recombinant His-tagged GMPS from parasitic protozoa.     

Adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate: A simultaneous protein binding assay

The adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate contents of microliter quantities of urine can be determined simultaneously by combining individual protein binding assays for the two nucleotides. ³²P-labeled adenosine 3′,5′-monophosphate is bound to a protein from bovine skeletal muscle, while a lobster muscle protein preparation is utilized for binding of ³H-labeled guanosine 3′,5′-monophosphate.     

Structural insight into the mechanism of substrate specificity and catalytic activity of an HD-domain phosphohydrolase: the 5'-deoxyribonucleotidase YfbR from Escherichia coli

HD-domain phosphohydrolases have nucleotidase and phosphodiesterase activities and play important roles in the metabolism of nucleotides and in signaling. We present three 2.1-Å-resolution crystal structures (one in the free state and two complexed with natural substrates) of an HD-domain phosphohydrolase, the Escherichia coli 5′-nucleotidase YfbR. The free-state structure of YfbR contains a large cavity accommodating the metal-coordinating HD motif (H33, H68, D69, and D137) and other conserved residues (R18, E72, and D77). Alanine scanning mutagenesis confirms that these residues are important for activity. Two structures of the catalytically inactive mutant E72A complexed with Co²⁺ and either thymidine-5′-monophosphate or 2′-deoxyriboadenosine-5′-monophosphate disclose the novel binding mode of deoxyribonucleotides in the active site. Residue R18 stabilizes the phosphate on the Co²⁺, and residue D77 forms a strong hydrogen bond critical for binding the ribose. The indole side chain of W19 is located close to the 2′-carbon atom of the deoxyribose moiety and is proposed to act as the selectivity switch for deoxyribonucleotide, which is supported by comparison to YfdR, another 5′-nucleotidase in E. coli. The nucleotide bases of both deoxyriboadenosine-5′-monophosphate and thymidine-5′-monophosphate make no specific hydrogen bonds with the protein, explaining the lack of nucleotide base selectivity. The YfbR E72A substrate complex structures also suggest a plausible single-step nucleophilic substitution mechanism. This is the first proposed molecular mechanism for an HD-domain phosphohydrolase based directly on substrate-bound crystal structures.     

Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii

New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage, suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of uridine 5′-monophosphate (UMP) and thymidine 5′-monophosphate (dTMP) pools, thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII-deficient mutants, restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within, or proximal to, the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition, several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development.     

Properties of xanthosine 5'-monophosphate-amidotransferase from Escherichia coli.

Xanthosine 5′-phosphate (XMP)-amidotransferase catalyzes the formation of guanosine 5′-phosphate (GMP) by aminating XMP with either the amide group of glutamine (amidotransferase) or ammonia (aminase). The glutamine-supported activity of the purified enzyme from Escherichia coli has been studied, and its properties have been compared with those of other amidotransferases. The following results have been obtained. (i) The glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), irreversibly inhibits the amidotransferase activity. A maximal rate of inhibition by DON is achieved in the presence of XMP, ATP, and Mg²⁺ with a pseudo-first-order rate constant of 0.276 min^?1. (ii) The total number of sulfhydryl groups is approximately 22 per dimer (126,000 M_r). In the absence of substrates, about 8 sulfhydryl groups per dimer are titratable with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), and in the presence of XMP, ATP, and Mg²⁺ an additional 6 cysteine residues per dimer become exposed. When the amidotransferase activity is inactivated by DON, only 8 sulfhydryl groups are titratable. DTNB, p-chloromercuribenzoate, and bromopyruvate all selectively inactivate the amidotransferase activity. These results are consistent with the hypothesis that cysteine residues which are exposed by the substrates are involved in the amidotransferase activity. (iii) The purified XMP amidotransferase contains a glutaminase activity which can be measured in the absence of GMP formation. The glutaminase activity requires XMP, Mg²⁺, and either psicofuranine, an analog of adenosine, or inorganic pyrophosphate (PP_i) and is inhibited by p-chloromercuribenzoate and DON. Maximal stimulation is observed with 100 μm psicofuranine or PP_i, and there is no further stimulation in the presence of both effectors. The apparent K_m is 31 μm with PP_i and 13 μm with psicofuranine; the V for glutamine hydrolysis is about 60% of the rate of the amidotransferase activity. The cooperative interactions between the binding of PP_i and psicofuranine have been confirmed. In the presence of 2.5 μm psicofuranine the K_m for PP_i is reduced 20-fold, but the maximal velocity is unchanged. Similarly, the apparent K_m for psicofuranine is reduced by low concentrations (10 μm) of PP_i. The “uncoupling” of the hydrolysis of glutamine from the amination of XMP is the basis for the reported inhibitory effects of psicofuranine and PP_i on the amidotransferase activity. (iv) Tris buffer selectively inhibits the XMP-amidotransferase activity by inhibiting the glutaminase activity. This inhibition is time dependent and reversible and may explain the previous reports on the inability of this enzyme to use glutamine as a substrate.     

Ammonia channeling in Plasmodium falciparum GMP synthetase: investigation by NMR spectroscopy and biochemical assays

GMP synthetase, a class I amidotransferase, catalyzes the last step of the purine biosynthetic pathway, where ammonia from glutamine is incorporated into xanthosine 5'-monophospate to yield guanosine 5'-monnophosphate as the main product. Combined biochemical, structural, and computational studies of glutamine amidotransferases have revealed the existence of physically separate active sites connected by molecular tunnels that efficiently transfer ammonia from the glutaminase site to the synthetase site. Here, we have investigated aspects of ammonia channeling in P. falciparum GMP synthetase using biochemical assays in conjunction with 15N-edited proton NMR spectroscopy. Our results suggest that (1) ammonia released from glutamine is not equilibrated with the external medium, (2) saturating concentrations of glutamine do not obliterate the incorporation of external ammonia into GMP, and (3) ammonia in the external medium can access the thioester intermediate when the ATPPase domain is bound to substrates. Further, mutation of Cys-102 to alanine confirmed its identity as the catalytic residue in the glutaminase domain, and ammonia-dependent assays on the mutant indicated glutamine to be a partial uncompetitive inhibitor of the enzyme.     

Substrate Specificity and Oligomerization of Human GMP Synthetase

Guanine monophosphate (GMP) synthetase is a bifunctional two-domain enzyme. The N-terminal glutaminase domain generates ammonia from glutamine and the C-terminal synthetase domain aminates xanthine monophosphate (XMP) to form GMP. Mammalian GMP synthetases (GMPSs) contain a 130-residue-long insert in the synthetase domain in comparison to bacterial proteins. We report here the structure of a eukaryotic GMPS. Substrate XMP was bound in the crystal structure of the human GMPS enzyme. XMP is bound to the synthetase domain and covered by a LID motif. The enzyme forms a dimer in the crystal structure with subunit orientations entirely different from the bacterial counterparts. The inserted sub-domain is shown to be involved in substrate binding and dimerization. Furthermore, the structural basis for XMP recognition is revealed as well as a potential allosteric site. Enzymes in the nucleotide metabolism typically display an increased activity in proliferating cells due to the increased need for nucleotides. Many drugs used as immunosuppressants and for treatment of cancer and viral diseases are indeed nucleobase- and nucleoside-based compounds, which are acting on or are activated by enzymes in this pathway. The information obtained from the crystal structure of human GMPS might therefore aid in understanding interactions of nucleoside-based drugs with GMPS and in structure-based design of GMPS-specific inhibitors.     

Regulatory Role of Adenine Nucleotides in the Biosynthesis of Guanosine 5′-Monophosphate

Derepression of the synthesis of inosine 5′-monophosphate (IMP) dehydrogenase and of xanthosine 5′-monophosphate (XMP) aminase in pur mutants of Escherichia coli which are blocked in the biosynthesis of adenine nucleotides and guanine nucleotides differs in two ways from derepression in pur mutants blocked exclusively in the biosynthesis of guanine nucleotides. (i) The maximal derepression is lower, and (ii) a sharp decrease in the specific activities of AMP dehydrogenase and XMP aminase occurs, following maximal derepression. From the in vivo and in vitro experiments described, it is shown that the lack of adenine nucleotides in derepressed pur mutants blocked in the biosynthesis of adenine and guanine nucleotides is responsible for these two phenomena. The adenine nucleotides are shown to play an important regulatory role in the biosynthesis of guanosine 5′-monophosphate (GMP). (i) They induce the syntheses of IMP dehydrogenase and XMP aminase. (The mechanism of induction may involve the expression of the gua operon.) (ii) They appear to have an activating function in IMP dehydrogenase and XMP aminase activity. The physiological importance of these regulatory characteristics of adenine nucleotides in the biosynthesis of GMP is discussed.     

Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA^Gln and Asn-tRNA^Asn by conversion of the misacylated Glu-tRNA^Gln and Asp-tRNA^Asn species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction.A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn²⁺ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn²⁺ binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA^Gln or Asn-tRNA^Asn.     

Structural modeling of RNase P RNA of the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5′ leader sequence of precursor tRNA (pre-tRNA). The RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure–function relationships of this multifaceted primordial ribozyme.     

Xanthosine-5'-phosphate amidotransferase from Escherichia coli.

The purified enzyme xanthosine-5'-monophosphate (XMP) aminase from Escherichia coli strain B-96 is shown to possess catalytic activity with either glutamine or ammonia as a substrate. This enzyme, which possesses identical subunits, has the following properties: (a) a pH optimum of 8.3 for both aminase and amidotransferase; (b) an apparent K-m for both glutamine and NH3 of 1 mM; (c) an amidotransferase that is approximately 2 times more active than the aminase; (d) a linear relationship between velocity and enzyme concentrationfor both activities; (e) inhibition of both activities by the glutamine analogue 6-diazo-5-oxo-L-norleucine, but the amidotransferase is more sensitive than the aminase; and (f) inhbiition of both activities by the adenosine analogue, psicofuranine, but again the amidotransferase activity is more sensitive than the aminase. The so-called XMP aminase from the E. coli mutant B-24-1 also has been examined in both crude extracts nad ammonium sulfate fractions and the following data have been obtained: (a) both preparations of enzyme contain aminase and amidotransferase activity; (b) both activities have the same substrate requirements; (c) the pH optima for both activities in the crude extract are identical with those found with the purified enzyme preparation; and (d) the amidotransferase activity in the crude extract and the ammonium sulfate fractions is 2- to 3-fold more active than the aminase. These data demonstrate that this enzyme from E. coli is not strictly a XMP aminase but is, in fact, an amidotransferase capable of utilizing either glutamine or NH3 as a substrate.     

MoImd4 mediates crosstalk between MoPdeH-cAMP signalling and purine metabolism to govern growth and pathogenicity in Magnaporthe oryzae

The high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance in the rice blast fungus Magnaporthe oryzae. To explore the underlying mechanism, we identified MoImd4 as an inosine-5′-monophosphate dehydrogenase (IMPDH) homologue that interacts with MoPdeH. Targeted deletion of MoIMD4 resulted in reduced de novo purine biosynthesis and growth, as well as attenuated pathogenicity, which were suppressed by exogenous xanthosine monophosphate (XMP). Treatment with mycophenolic acid (MPA), which specifically inhibits MoImd4 activity, resulted in reduced growth and virulence attenuation. Intriguingly, further analysis showed that MoImd4 promotes the phosphodiesterase activity of MoPdeH, thereby decreasing intracellular cAMP levels, and MoPdeH also promotes the IMPDH activity of MoImd4. Our studies revealed the presence of a novel crosstalk between cAMP regulation and purine biosynthesis in M. oryzae, and indicated that such a link is also important in the pathogenesis of M. oryzae.     

Mycobacterium tuberculosis IMPDH in Complexes with Substrates,Products and Antitubercular Compounds

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD⁺ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD⁺ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.     

Hyperthermophilic archaeal prefoldin shows refolding activity at low temperature

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself. In this study, we have demonstrated for the first time that a prefoldin from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhPFD), exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures. Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein-folding seems to occur through binding and release from PhPFD by virtue of the weak affinity. Our results also imply that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak.     

Crystal structures of Streptococcus mutans 2'-deoxycytidylate deaminase and its complex with substrate analog and allosteric regulator dCTP x Mg2+

2′-Deoxycytidylate deaminase [or deoxycytidine-5′-monophosphate (dCMP) deaminase, dCD] catalyzes the deamination of dCMP to deoxyuridine-5′-monophosphate to provide the main nucleotide substrate for thymidylate synthase, which is important in DNA synthesis. The activity of this homohexameric enzyme is allosterically regulated by deoxycytidine-5′-triphosphate (dCTP) as an activator and by deoxythymidine-5′-triphosphate as an inhibitor. In this article, we report the crystal structures of dCMP deaminase from Streptococcus mutans and its complex with dCTP and an intermediate analog at resolutions of 3.0 and 1.66 Å. The protein forms a hexamer composed of subunits adopting a three-layer α/β/α sandwich fold. The positive allosteric regulator dCTP mainly binds at the interface between two monomers in a molar ratio of 1:1 and rearranges the neighboring interaction networks. Structural comparisons and sequence alignments revealed that dCMP deaminase from Streptococcus mutans belongs to the cytidine deaminase superfamily, wherein the proteins exhibit a similar catalytic mechanism. In addition to the two conserved motifs involved in the binding of Zn^2 +, a new conserved motif, (G⁴³YNG⁴⁶), related to the binding of dCTP was also identified. N-terminal Arg4, a key residue located between two monomers, binds strongly to the γ phosphate group of dCTP. The regulation signal was transmitted by Arg4 from the allosteric site to the active site via modifications in the interactions at the interface where the substrate-binding pocket was involved and the relocations of Arg26, His65, Tyr120, and Arg121 to envelope the active site in order to stabilize substrate binding in the complex. Based on the enzyme-regulator complex structure observed in this study, we propose an allosteric mechanism for dCD regulation.     

Control of feedback repression of the enzymes of purine biosynthesis in Aerobacter aerogenes

Studies have been made of the regulation of the synthesis of six purine biosynthetic enzymes: P-ribosyl-PP amidotransferase (I), P-ribosyl glycinamide synthetase (II), P-ribosyl formyl glycinamide amidotransferase (IV), adenylosuccinate lyase (VIII-IIA), adenylosuccinate synthetase (IA), and IMP dehydrogenase (IG). Wild type Aerobacter aerogenes and two purine requiring mutants derived from it, were grown with limiting or excess adenine or guanine, cell extracts prepared, and enzyme activities measured.     

Ligand binding induces an ammonia channel in 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE

PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. The PhzE monomer contains both a chorismate-converting menaquinone, siderophore, tryptophan biosynthesis (MST) and a type 1 glutamine amidotransferase (GATase1) domain connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligand-free open and ligand-bound closed conformation at 2.9 and 2.1 Å resolution, respectively. PhzE arranges in an intertwined dimer such that the GATase1 domain of one chain provides NH₃ to the MST domain of the other. This quaternary structure was confirmed by small angle x-ray scattering. Binding of chorismic acid, which was found converted to benzoate and pyruvate in the MST active centers of the closed form, leads to structural rearrangements that establish an ammonia transport channel approximately 25 Å in length within each of the two MST/GATase1 functional units of the dimer. The assignment of PhzE as an ADIC synthase was confirmed by mass spectrometric analysis of the product, which was also visualized at 1.9 Å resolution by trapping in crystals of an inactive mutant of PhzD, an isochorismatase that catalyzes the subsequent step in phenazine biosynthesis. Unlike in some of the related anthranilate synthases, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1 active center was identified as zinc, and it was demonstrated that Zn²⁺, Mn²⁺, and Ni²⁺ reduce the activity of PhzE.     

拟南芥GATL12基因影响叶绿体的形成

谷氨酰胺氨基转移酶（GATase）能够将谷氨酰胺上的氨基基团转移到底物上形成新的一碳氮基团。GATase有两种类型,即Class-I（trpG型）和Class-II（purF型）。拟南芥（Arabidopsis thaliana）基因组中有13个基因编码Class-I类似蛋白（GATLs）,其生物学功能尚不清楚。首先分离到拟南芥GATL12基因的2个T-DNA插入突变体,分别命名为gatl12-1和gatl12-2。然后观察发现在这2个突变体的杂合植株中,大部分植株的胚珠发育到第8天时,由于叶绿体的积累而呈现绿色,其余植株（约有25%）的胚珠为白色。将从杂合突变体植株上收获的种子播种在1/2MS培养基上,有25%的幼苗发育成黄化苗。经PCR检测,这些黄化苗为GATL12的纯合突变体,RT-PCR法在黄化苗中检测不到GATL12基因的转录本。电镜观察表明,突变体中的叶绿体不能正常发育。上述结果表明,GATL12基因在拟南芥的叶绿体发育过程中具有重要作用。     

The Effects of Removing the GAT Domain from E. coli GMP Synthetase

E. coli GMP synthetase (GMPS) catalyzes the conversion of XMP to GMP. Ammonia, generated in the amino-terminal glutamine amidotransferase (GAT) domain, is transferred by an unknown mechanism to the ATP-pyrophosphatase (ATPP) domain, where it attacks a highly reactive adenyl-XMP intermediate, leading to GMP formation. To study the structural requirements for the activity of E. coli GMPS, we used PCR to generate a protein expression construct that contains the ATPP domain as well as the predicted dimerization domain (DD). The ATPP/DD protein is active in solution, utilizing NH₄⁺ as an NH₃ donor. Size-exclusion chromatography demonstrates a dimeric mass for the ATPP/ DD protein, providing the first evidence in solution for the structural organization of the intact GMPS. Kinetic characterization of the ATPP/DD domain protein provides evidence that the presence of the GAT domain can regulate the activity of the ATPP domain.     

Nucleotide Binding States of Subunit A of the A-ATP Synthase and the Implication of P-Loop Switch in Evolution

The crystal structures of the nucleotide-empty (A_E), 5′-adenylyl-β,γ-imidodiphosphate (A_PNP)-bound, and ADP (A_DP)-bound forms of the catalytic A subunit of the energy producer A₁A_O ATP synthase from Pyrococcus horikoshii OT3 have been solved at 2.47 Å and 2.4 Å resolutions. The structures provide novel features of nucleotide binding and depict the residues involved in the catalysis of the A subunit. In the A_E form, the phosphate analog SO₄²⁻ binds, via a water molecule, to the phosphate binding loop (P-loop) residue Ser238, which is also involved in the phosphate binding of ADP and 5′-adenylyl-β,γ-imidodiphosphate. Together with amino acids Gly234 and Phe236, the serine residue stabilizes the arched P-loop conformation of subunit A, as shown by the 2.4-Å structure of the mutant protein S238A in which the P-loop flips into a relaxed state, comparable to the one in catalytic β subunits of F₁F_O ATP synthases. Superposition of the existing P-loop structures of ATPases emphasizes the unique P-loop in subunit A, which is also discussed in the light of an evolutionary P-loop switch in related A₁A_O ATP synthases, F₁F_O ATP synthases, and vacuolar ATPases and implicates diverse catalytic mechanisms inside these biological motors.