Substrate-induced conformational changes in Plasmodium falciparum guanosine monophosphate synthetase

GMP synthetase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the nucleotide xanthosine 5'-monophosphate (XMP) to form guanosine 5'-monophosphate (GMP). Functional coordination of domains in glutamine amidotransferases leads to upregulation of glutamine hydrolysis in the presence of acceptor substrates and is a common feature in this class of enzymes. We have shown earlier that binding of substrates to the acceptor domain of Plasmodium falciparum GMP synthetase (PfGMPS) leads to enhancement in both glutaminase activity and rate of glutaminase inactivation, by the irreversible inhibitors acivicin and diazo-oxonorleucine [Bhat JY et al. (2008) Biochem J409, 263-273], a process that must be driven by conformational alterations. In this paper, through the combined use of biochemical assays, optical spectroscopy and mass spectrometry, we demonstrate that PfGMPS undergoes conformational transitions upon binding of substrates to the acceptor domain. Limited proteolysis and hydrogen-deuterium exchange in conjunction with mass spectrometry unveil region-specific conformational changes in the ATP + XMP bound state of PfGMPS. Decreased accessibility of R294 and K428 residues to trypsin in the ATP pyrophosphatase domain and reduced deuterium incorporation in the 143-155 region, pertaining to the glutaminase domain, suggest that in PfGMPS ligand-induced conformational changes are not only local but also transmitted over a long range across the domains. Overall, these results provide a detailed understanding of the substrate-induced changes in PfGMPS that could be essential for the overall catalytic process.     

Crystal Structure of the ATPPase Subunit and Its Substrate-Dependent Association with the GATase Subunit: A Novel Regulatory Mechanism for a Two-Subunit-Type GMP Synthetase from Pyrococcus horikoshii OT3

Guanosine 5′-monophosphate synthetase(s) (GMPS) catalyzes the final step of the de novo synthetic pathway of purine nucleotides. GMPS consists of two functional units that are present as domains or subunits: glutamine amidotransferase (GATase) and ATP pyrophosphatase (ATPPase). GATase hydrolyzes glutamine to yield glutamate and ammonia, while ATPPase utilizes ammonia to convert adenyl xanthosine 5′-monophosphate (adenyl-XMP) into guanosine 5′-monophosphate. Here we report the crystal structure of PH-ATPPase (the ATPPase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon Pyrococcus horikoshii OT3). PH-ATPPase consists of two domains (N-domain and C-domain) and exists as a homodimer in the crystal and in solution. The N-domain contains an ATP-binding platform called P-loop, whereas the C-domain contains the xanthosine 5'-monophosphate (XMP)-binding site and also contributes to homodimerization. We have also demonstrated that PH-GATase (the glutamine amidotransferase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon P. horikoshii OT3) alone is inactive, and that all substrates of PH-ATPPase except for ammonia (Mg²⁺, ATP and XMP) are required to stabilize the active complex of PH-ATPPase and PH-GATase subunits.     

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.     

Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain

Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PP(i), and glutamate. XMP, NH(4)(+), and Mg(2+) displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH(3) and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PP(i) is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family.     

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.     

Positional isotope exchange and kinetic experiments with Escherichia coli guanosine-5'-monophosphate synthetase

The kinetic mechanism of Escherichia coli guanosine-5'-monophosphate synthetase has been determined by utilizing initial velocity kinetic patterns and positional isotope exchange experiments. The initial velocity patterns of MgATP, XMP, and either NH3 or glutamine (as nitrogen source) were consistent with the ordered addition of MgATP followed by XMP and then NH3. The enzyme catalyzes the exchange of 18O from the beta-nonbridge positions of [beta,beta,beta gamma,gamma,gamma,gamma-18O6]ATP into the alpha beta-bridge position only in the presence of XMP and Mg2+. The exchange reaction did not require NH3. The isotope exchange reaction increased as the XMP concentration increased and then decreased at saturating levels of XMP. These results also support the ordered addition of MgATP followed by XMP. GMP synthetase catalyzes the hydrolysis of ATP to AMP and PPi along with an ATP/PPi exchange reaction in the absence of NH3. These data taken together support a mechanism in which the initial step in the enzymatic reaction involves formation of an adenyl-XMP intermediate. Psicofuranine, an irreversible inhibitor of the enzyme, acts by preventing the release or further reaction of adenyl-XMP with H2O or NH3 but does not suppress the isotope exchange or ATP/PPi exchange reactions. GMP synthetase has also been shown to require a free divalent cation for full activity. When Ca2+ replaces Mg2+ in the reaction, the positional isotope exchange reaction is enhanced but the reaction with NH3 to form GMP is greatly suppressed.     

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.     

Preliminary x-ray analysis of Escherichia coli GMP synthetase: Determination of anomalous scattering factors for a cysteinyl mercury derivative

We have initiated a project to determine the three-dimensional structure of GMP synthetase (GMPS) from Escherichia coli. GMPS catalyzes the conversion of XMP to GMP in the final step of de novo guanine nucleotide biosynthesis, and is a member of the glutamine amidotransferase family: a group of enzymes responsible for the assimilation of nitrogen into compounds such as amino acids, purine and pyrimidine bases, amino sugars, and antibiotics. The E. coli guaA gene encoding GMPS was cloned into a tac expression vector, overexpressed, and its gene product purified. Conditions for the growth of protein crystals were developed using recombinant GMPS in the presence of MgCl₂, ATP, and XMP. The crystals are monoclinic, space group P2₁, with cell parameters of a = 156.0 Å, b = 102.0 Å, c = 78.8 Å, β= 96.7°. Diffraction data to 2.8 Å spacings were collected on a Xuong-Hamlin area detector with an overall R_sym of 5.2%. Both the volume of the unit cell and the peaks in the self-rotation function are consistent with one GMPS tetramer of D₂ symmetry in the crystallographic asymmetric unit. Previously, GMPS has been observed only as a dimer in solution. GMPS was covalently modified with p-chloromercuribenzylsulfonic acid (PCMBS), and its X-ray fluorescence spectrum was measured through the L_III absorption edge of mercury Anomalous scattering factors for cysteinyl mercury were derived from this spectrum, and the feasibility of structure determination by multi-wavelength anomalous diffraction was evaluated. The optimal MAD dispersive signal is 4.5% of |F|, and the optimal MAD Bijvoet signal is 7.5% of |F| at a concentration of approximately 1 mercury per 10-kDa protein. The anomalous scattering factors tabulated here should be transferable to cysteinyl mercury in other proteins. © 1994 John Wiley & Sons, Inc.     

Mechanism for acivicin inactivation of triad glutamine amidotransferases

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.     

The synthesis of pABA: Coupling between the glutamine amidotransferase and aminodeoxychorismate synthase domains of the bifunctional aminodeoxychorismate synthase from Arabidopsis thaliana

Aminodeoxychorismate (ADC) synthase in plants is a bifunctional enzyme containing glutamine amidotransferase (GAT) and ADC synthase (ADCS) domains. The GAT domain releases NH₃ from glutamine and the ADCS domain uses NH₃ to aminate chorismate. This enzyme is involved in folate (vitamin B9) biosynthesis. We produced a stable recombinant GAT–ADCS from Arabidopsis. Its kinetic properties were characterized, and activities and coupling of the two domains assessed. Both domains could operate independently, but not at their optimal capacities. When coupled, the activity of one domain modified the catalytic properties of the other. The GAT activity increased in the presence of chorismate, an activation process that probably involved conformational changes. The ADCS catalytic efficiency was 10⁴ fold higher with glutamine than with NH₄Cl, indicating that NH₃ released from glutamine and used for ADC synthesis did not equilibrate with the external medium. We observed that the GAT activity was always higher than that of ADCS, the excess of NH₃ being released in the external medium. In addition, we observed that ADC accumulation retro-inhibited ADCS activity. Altogether, these results indicate that channeling of NH₃ between the two domains and/or amination of chorismate are the limiting step of the whole process, and that ADC cannot accumulate.     

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.     

Mutation analysis of carbamoyl phosphate synthetase: Does the structurally conserved glutamine amidotransferase triad act as a functional dyad?

Evolutionarily conserved triad glutamine amidotransferase (GAT) domains catalyze the cleavage of glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. Whereas a conserved hydrolytic triad (Cys/His/Glu) is observed in the solved GAT structures, the specificity pocket for glutamine is not apparent, presumably because its formation is dependent on the conformational change that couples acceptor availability to a greatly increased rate of glutamine cleavage. In Escherichia coli carbamoyl phosphate synthetase (eCPS), one of the best characterized triad GAT members, the Cys269 and His353 triad residues are essential for glutamine hydrolysis, whereas Glu355 is not critical for eCPS activity. To further define the glutamine-binding pocket and possibly identify an alternative member of the catalytic triad that is situated for this role in the coupled conformation, we have analyzed mutations at Gln310, Asn311, Asp334, and Gln351, four conserved, but not yet analyzed residues that might potentially function as the third triad member. Alanine substitution of Gln351, Asn311, and Gln310 yielded respective K(m) increases of 145, 27, and 15, suggesting that Gln351 plays a key role in glutamine binding in the coupled conformation, and that Asn311 and Gln310 make less significant contributions. None of the mutant k (cat) values varied significantly from those for wild-type eCPS. Combined with previously reported data on other conserved eCPS residues, these results strongly suggest that Cys269 and His353 function as a catalytic dyad in the GAT site of eCPS.     

Gain of glutaminase function in mutants of the ammonia-specific frog carbamoyl phosphate synthetase

Depending on their physiological role, carbamoyl phosphate synthetases (CPSs) use either glutamine or free ammonia as the nitrogen donor for carbamoyl phosphate synthesis. Sequence analysis of known CPSs indicates that, regardless of whether they are ammonia- or glutamine-specific, all CPSs contain the structural equivalent of a triad-type glutamine amidotransferase (GAT) domain. In ammonia-specific CPSs, such as those of rat or human, the catalytic inactivity of the GAT domain can be rationalized by the substitution of the Triad cysteine residue by serine (1). The ammonia-specific CPS of Rana catesbeiana (fCPS) presents an interesting anomaly in that, despite its retention of the entire catalytic triad (2) and almost all other residues conserved in Triad GATs, it is unable to utilize glutamine as a nitrogen-donating substrate (3). Based on our earlier work with the glutamine-utilizing E. coli CPS (eCPS), we have targeted residues Lys258 and Glu261 in the fCPS GAT domain as critical for preventing GAT function. Previously we have shown that substitution of the corresponding residues in eCPS by their fCPS counterparts (Leu --> Lys and Gln --> Glu) resulted in complete loss of GAT function in eCPS (3). To examine the role of these residues in the fCPS GAT component, we have cloned the full-length fCPS gene from R. catesbeiana liver. Here we report the first heterologous expression of an ammonia-specific CPS and show that a single mutation of the frog enzyme, K258L, yields a gain of glutaminase function.     

The C-terminal domain of HPII catalase is a member of the type I glutamine amidotransferase superfamily

Discovering distant evolutionary relationships between proteins requires detecting subtle similarities. Here we use a combination of sequence and structure analysis to show that the C-terminal domain of Escherichia coli HPII catalase with available spatial structure is a divergent member of the type I glutamine amidotransferase (GAT) superfamily. GAT-containing proteins include many biosynthetic enzymes such as E. coli carbamoyl phosphate synthetase and anthranilate synthase. Typical GAT domains have Rossmann fold-like topology and possess a catalytic triad similar to that of proteases. The C-terminal domain of HPII catalase has the GAT Rossmann fold but lacks the triad and therefore loses enzymatic activity. In addition, we detect significant sequence similarity between thiJ domains, some of which are known to have protease activity, and typical GAT proteins. Evolutionary tree analysis of the entire GAT superfamily indicates that the HPII catalase is more closely related to thiJ domains than to classical GAT domains and is likely to have evolved from a thiJ-like protein. This work illustrates the strength of sequence-based profile analysis techniques coupled with structural superpositions in developing an evolutionarily relevant classification of protein structures. Proteins 2001;42:230-236.     

Agaricus bisporus and Coprinus bilanatus TRP2 genes are tri-functional with conserved intron and domain organisations

Cloned homobasidiomycete TRP2 genes for Agaricus bisporus and Coprinus bilanatus were sequence-characterised. Both genes encode tri-functional proteins with activity domains for glutamine amidotransferase (GAT; G domain), indole glycerol phosphate synthase (InGP; C domain) and phosphoribosyl anthranilate isomerase (F domain). A conserved intron disrupts the GAT-coding sequence in both genes. Consensus amino acid (aa) signatures were identified for GAT and InGP, but in the latter 15-aa signature, one residue did not fit the previously defined consensus. Protein architecture and parsimony analysis with analogous proteins indicate domain organisation (NH(2)-G-C-F-COOH) was as for other filamentous fungi. The data do not support earlier suggestions that the three activity domains are detached in A. bisporus.     

Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii

Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (Plasmodium, Babesia, Toxoplasma) or zoomastigina (Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase.     

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。     

GMP synthase is essential for viability and infectivity of Trypanosoma brucei despite a redundant purine salvage pathway

The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine‐5′‐monophosphate (GMP) formation: conversion from xanthosine‐5′‐monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine‐guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 μM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of human African trypanosomiasis. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche.     

The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.