An anthranilate synthase of the extreme aminase type in a species of blue-green bacteria (algae)

Anthranilate synthase of Agmenellum quadruplicatum, a unicellular species of blue-green bacteria, consists of two nonidentical subunits. A 72,000 dalton protein has aminase activity but is incapable of reaction with glutamine (amidotransferase) unless a second protein (18,000 molecular weight) is present. The small subunit was first detected through its ability to complement a partially purified aminase subunit from Bacillus subtilis to produce a hybrid complex capable of amidotransferase function. Conditions for the function of the heterologous complex were less stringent than for the homologous A. quadruplicatum complex. A reducing agent such as dithiothreitol stabilizes the A. quadruplicatum aminase subunit and is obligatory for amidotransferase function. L-Tryptophan feedback inhibits both the aminase and amidotransferase reactions of anthranilate synthase; Ki values of 6 X 10(-8) M for the amidotransferase activity and 2 X 10(-6) M for the aminase activity were obtained. The Km value calculated for ammonia (2.2 mM) was more favorable than the Km value glutamine (13 mM). Likewise, the Vmax of anthranilate synthase was greater with ammonia than with glutamine. Starvation of a tryptophan auxotroph results in a threefold derepression of the aminase subunit, but no corresponding increase in the small 18,000 M subunit occurs. While microbial anthranilate synthase complexes are remarkably similar overall, the relatively good aminase activity of the A. quadruplicatum enzyme may be of physiological significance in nature.     

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.     

Characterization and regulation of p-aminobenzoic acid synthase from Streptomyces griseus

p-Aminobenzoic acid synthase (PABA synthase) of Streptomyces griseus catalyses the conversion of chorismic acid to p-aminobenzoic acid (PABA), a precursor of the aromatic p-aminoacetophenone moiety of candicidin, a polyene macrolide antibiotic. This enzyme uses glutamine or ammonia as amino donors for PABA formation. Enzyme extracts converted [14C]chorismic acid to labelled PABA. PABA synthase was present in S. griseus IMRU 3570 only during the antibiotic producing phase. No detectable levels of the enzyme were found in cell-free extracts of nonproducing mutants of S. griseus obtained after UV mutagenesis. PABA synthase activity was found also in Streptomyces coelicolor var. aminophilus, producer of the polyene macrolide antibiotic fungimycin, but it was not present in extracts of several other streptomycetes that do not produce aromatic polyene macrolide antibiotics. PABA synthase (amidotransferase) activity was partially purified by DEAE-Bio-gel and Sephacryl S-200 filtrations. The estimated molecular weight was 50000. PABA synthase was repressed by aromatic amino acids and PABA but not by anthranilic acid. Inorganic phosphate strongly repressed but did not inhibit PABA synthase activity.     

Regulation of a common amidotransferase subunit.

In Bacillus subtilis the trpX locus specifies a glutamine-binding protein designated subunit X, which forms a complex with subunit E to constitute the anthranilate synthase enzyme aggregate (EX) and subunit A to constitute the p-aminobenzoate synthase enzyme aggregate (AX). Subunit X confers upon these enzyme complexes the ability to utilize glutamine as a substrate. The trpX locus has been examined to determine its map position and control. (i) The trpX locus was found to be cotransformed with the lysS and pabA loci. The results of three-factor transformation analyses suggest the following order of these markers: lysS-sul-trpX-pabA. (ii) Mutation to constitutivity of the tryptophan operon resulted in a 50- to 60-fold increase in the level of subunit X when the mutant contained functional trE and abA gene products; however, in the absence of subunit E there was only a 4- to 5-fold increase in the glutamine-binding protein. (iii) Formation of subunit X was derepressed under conditions that allow for the derepression of the trpE and/or pabA loci. (iv) Subunit X synthesis was derepressed to a greater extent in mutants that contain a functional trpE gene product than in mutants that contain a nonsense mutation in the trpE locus. These results are consistent with the hypothesis that the trpE and pabA gene products affect the expression and control of the trpX locus.     

Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.     

Subunit interactions and glutamine utilization by Escherichia coli imidazole glycerol phosphate synthase

A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimeric Escherichia coli imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however, k(cat)/K(m) for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites.     

Homologous and Hybrid Complexes of Anthranilate Synthase from Bacillus Species

The subunits of anthranilate synthase were separated and partially purified by Sephadex G-100 gel filtration from the following six species of Bacillus: Bacillus subtilis, Bacillus licheniformis, Bacillus alvei, Bacillus coagulans, Bacillus pumilus, and Bacillus mascerans. Our data suggest that the enzyme from B. alvei is unique among these species. First, the anthranilate synthase complexes are readily dissociated during gel filtration in the absence of glutamine into a large component (aminotransferase), subunit E, and a small component subunit X (glutamine-binding protein), whereas a higher salt concentration is required to dissociate the complex from B. alvei. Second, the aminotransferase activity from all six species is stimulated by glycerol and inhibited by tryptophan; however, only the large component from B. alvei is stimulated by 2-mercaptoethanol. Finally, the large component can be titrated with the small component to yield a complex which can utilize glutamine as a substrate (amidotransferase). The homologous complexes have an amidotransferase to aminotransferase ratio of 1.4 to 2.3, but the B. alvei complex has a ratio of 0.9. Except for complexes that involve the large component from B. alvei, hybrid complexes can be formed which have ratios as good as the homologous complexes. These data are consistent with the hypothesis that B. alvei is unique among the bacilli with respect to some enzymes in the aromatic amino acid biosynthetic pathway.     

Crystal Structures Capture Three States in the Catalytic Cycle of a Pyridoxal Phosphate (PLP) Synthase

PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5′-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.     

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli.

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the Km values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.     

Constitutively active glutaminase variants provide insights into the activation mechanism of anthranilate synthase

The glutamine amidotransferase (GATase) family comprises enzyme complexes which consist of glutaminase and synthase subunits that catalyze in a concerted reaction the incorporation of nitrogen within various metabolic pathways. An important feature of GATases is the strong stimulation of glutaminase activity by the associated synthase. To understand the mechanism of this tight activity regulation, we probed by site-directed mutagenesis four residues of the glutaminase subunit TrpG from anthranilate synthase that are located between the catalytic Cys-His-Glu triad and the synthase subunit TrpE. In order to minimize structural perturbations induced by the introduced exchanges, the amino acids from TrpG were substituted with the corresponding residues of the closely related glutaminase HisH from imidazole glycerol phosphate synthase. Steady-state kinetic characterization showed that, in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyzed glutamine in the absence of TrpE. A reaction assay performed with hydroxylamine as a stronger nucleophile replacing water and a filter assay with radiolabeled glutamine indicated that the formation of the thioester intermediate is the rate-limiting step of constitutive glutamine hydrolysis. Molecular dynamics simulations with wild-type TrpG and constitutively active TrpG variants suggest that the introduced amino acid exchanges result in a distance reduction between the active site Cys-His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain. We propose that native TrpG in the anthranilate synthase complex is activated by a similar mechanism.     

Anthranilate synthase of Neurospora crassa: reaction and labeling with glutamine analogs

The multifunctional enzyme complex, anthranilate synthase from Neurospora crassa, irreversibly loses its glutamine-dependent anthranilate synthase activity on exposure to the reactive glutamine analogs DON and azaserine. Inactivation depends on the presence of the substrate chorismate, is enhanced by the cofactor Mg⁺², and is antagonized by glutamine. Inactivation correlates well with the incorporation of [¹⁴C]DON into the protein with modification localized to the β subunit (M_r 84,000) of the complex, demonstrating directly that the β subunit provides the glutamine binding site for the glutamine-dependent anthranilate synthase reaction. The slower and less extensive loss of ammonia-dependent anthranilate synthase activity indicates that maximum expression of the ammonia-dependent anthranilate synthase activity by the α subunit also depends on the interaction with an active glutamine amidotransferase domain of the β subunit.     

Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.     

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.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: I. Sequence of trpG encoding the glutamine amidotransferase subunit

We have determined the DNA sequence of the distal 148 codons of trpE and all of trpG in Pseudomonas aeruginosa. These genes encode, respectively, the large and small (glutamine amidotransferase) subunits of anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. The sequenced region of trpE is homologous with the distal portion of E. coli and Bacillus subtilis trpE, whereas the trpG sequence is homologous to the glutamine amidotransferase subunit genes of a number of bacterial and fungal anthranilate synthases. The two coding sequences overlap by 23 bp. Codon usage in these Pseudomonas genes shows a marked preference for codons ending in G or C, thereby resembling that of trpB, trpA, and several other chromosomal loci from this species and others with a high G + C content in their DNA. The deduced amino acid sequence for the P. aeruginosa trpG gene product differs to a surprising extent from the directly determined amino acid sequence of the glutamine amidotransferase subunit of P. putida anthranilate synthase (Kawamura et al. 1978). This suggests that these two proteins are encoded by loci that duplicated much earlier in the phylogeny of these organisms but have recently assumed the same function. We have also determined 490 bp of DNA sequence distal to trpG but have not ascertained the function of this segment, though it is rich in dyad symmetries.      

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.     

Modification of Serratia marcescens anthranilate synthase with pyridoxal 5′-phosphate

The glutamine-dependent activity of Serratia marcescens anthranilate synthase was inactivated by pyridoxal 5′-phosphate and sodium cyanide. The reaction was specific in that the ammonia-dependent activity of the enzyme was unaffected. The inactivation was stable to dilution or dialysis but was reversed by dithiothreitol. The enzyme contains dissimilar subunits designated anthranilate synthase components I (AS I) and II (AS II). Incorporation of [¹⁴C]NaCN demonstrates that modification was limited to one to two residues per AS I · AS II protomer. An active site cysteine is involved in the glutamine-dependent activity. Modification by pyridoxal 5′-phosphate and NaCN blocked affinity labeling of the active site cysteine by the glutamine analog 6-diazo-5-oxo-l-norleucine and reduced alkylation of the active site cysteine by iodoacetamide. These results suggest modification is at the glutamine active site. Initial modification by iodoacetamide did not prevent pyridoxal 5′-phosphate-dependent incorporation of ¹⁴CN showing that the pyridoxal 5′-phosphate modification did not involve the essential cysteinyl residue. These results suggest that modification of a lysyl residue in the glutamine active site of anthranilate synthase reduces the reactivity of the essential cysteinyl residue resulting in the loss of the amidotransferase activity.     

Alternative substrates for wild-type and L109A E. coli CTP synthases: kinetic evidence for a constricted ammonia tunnel.

Cytidine 5'-triphosphate (CTP) synthase catalyses the ATP-dependent formation of CTP from uridine 5'-triphosphate using either NH(3) or l-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH(3) that is generated is transferred via an NH(3) tunnel [Endrizzi, J.A., Kim, H., Anderson, P.M. & Baldwin, E.P. (2004) Biochemistry43, 6447-6463] to the active site of the N-terminal synthase domain where the amination reaction occurs. Replacement of Leu109 by alanine in Escherichia coli CTP synthase causes an uncoupling of glutamine hydrolysis and glutamine-dependent CTP formation [Iyengar, A. & Bearne, S.L. (2003) Biochem. J.369, 497-507]. To test our hypothesis that L109A CTP synthase has a constricted or a leaky NH(3) tunnel, we examined the ability of wild-type and L109A CTP synthases to utilize NH(3), NH(2)OH, and NH(2)NH(2) as exogenous substrates, and as nascent substrates generated via the hydrolysis of glutamine, gamma-glutamyl hydroxamate, and gamma-glutamyl hydrazide, respectively. We show that the uncoupling of the hydrolysis of gamma-glutamyl hydroxamate and nascent NH(2)OH production from N(4)-hydroxy-CTP formation is more pronounced with the L109A enzyme, relative to the wild-type CTP synthase. These results suggest that the NH(3) tunnel of L109A, in the presence of bound allosteric effector guanosine 5'-triphosphate, is not leaky but contains a constriction that discriminates between NH(3) and NH(2)OH on the basis of size.     

Structure of imidazole glycerol phosphate synthase from Thermus thermophilus HB8: open-closed conformational change and ammonia tunneling

Imidazole glycerol phosphate synthase (IGPs) catalyzes the fifth step in the histidine biosynthetic pathway located at the branch point to de novo purine biosynthesis. IGPs is a multienzyme comprising glutaminase and synthase subunits. The glutaminase activity, which hydrolyzes glutamine to give ammonia, is coupled with substrate binding to the synthase subunit. The three-dimensional structure of the IGPs from Thermus thermophilus HB8 has been determined at 2.3 A resolution, and compared with the previously determined structures for the yeast and Thermotoga maritima enzymes. The structure of each subunit is similar to that of the corresponding domain in the yeast enzyme or subunit in the T. maritima enzyme. However, the overall structure is significantly different from the yeast and T. maritima enzymes, indicating that IGPs may change the relative orientation between the two subunits and close the glutaminase site upon glutamine binding. The putative ammonia tunnel, which carries nascent ammonia from glutaminase to the synthase site, has a closed gate comprising a cyclic salt bridge formed by four charged residues of the synthase subunit. The side chain of Lys100 in the cyclic salt bridge might change its side chain direction to form new interactions with the main chain carbonyl group of glutamine from the synthase subunit and the hydoxyl group of tyrosine from the glutaminase subunit, resulting in the opening of the gate for ammonia transfer.     

Rapid Regulation of an Anthranilate Synthase Aggregate by Hysteresis

The anthranilate synthase aggregate from Bacillus subtilis is composed of two nonidentical subunits, denoted E and X, which are readily associated or dissociated. A complex of subunit E and X can utilize glutamine or ammonia as substrates in the formation of anthranilate. Partially purified subunit E is capable of using only ammonia as the amide donor in the anthranilate synthase reaction. The stability of the EX complex is strongly influenced by glutamine and by the concentrations of the subunits. Glutamine stabilizes the aggregate as a molecular species in which the velocity of the glutamine-reactive anthranilate synthase is a linear function of protein concentration. In the absence of glutamine the aggregate is readily dissociated following dilution of the extract; that is, velocity concaves upward as a function of increasing protein concentration. Reassociation of the EX complex is characterized by a velocity lag (or hysteretic response) before steady-state velocity for the glutamine-reactive anthranilate synthase is reached. We propose that association and dissociation of the anthranilate synthase aggregate may be physiologically significant and provide a control mechanism whereby repression or derepression causes disproportionate losses or gains in activity by virtue of protein-protein interactions between subunits E and X.