Molecular dynamics simulation of the interaction between the complex iron-sulfur flavoprotein glutamate synthase and its substrates

Glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of L-glutamine amide group to the C2 carbon of 2-oxoglutarate yielding two molecules of L-glutamate. Molecular dynamics calculations in explicit solvent were carried out to gain insight into the conformational flexibility of GltS and into the role played by the enzyme substrates in regulating the catalytic cycle. We have modelled the free (unliganded) form of Azospirillum brasilense GltS alpha subunit and the structure of the reduced enzyme in complex with the L-glutamine and 2-oxoglutarate substrates starting from the crystallographically determined coordinates of the GltS alpha subunit in complex with L-methionine sulphone and 2-oxoglutarate. The present 4-ns molecular dynamics calculations reveal that the GltS glutaminase site may exist in a catalytically inactive conformation unable to bind glutamine, and in a catalytically competent conformation, which is stabilized by the glutamine substrate. Substrates binding also induce (1) closure of the loop formed by residues 263-271 with partial shielding of the glutaminase site from solvent, and (2) widening of the ammonia tunnel entrance at the glutaminase end to allow for ammonia diffusion toward the synthase site. The Q-loop of glutamate synthase, which acts as an active site lid in other amidotransferases, seems to maintain an open conformation. Finally, binding of L-methionine sulfone, a glutamine analog that mimics the tetrahedral transient species occurring during its hydrolysis, causes a coordinated rigid-body motion of segments of the glutaminase domain that results in the inactive conformation observed in the crystal structure of GltS alpha subunit.     

Mechanistic studies on Azospirillum brasilense glutamate synthase.

The reaction mechanism of Azospirillum brasilense glutamate synthase has been investigated by several approaches. 15N nuclear magnetic resonance studies demonstrate that the amide nitrogen of glutamine is reductively transferred to 2-oxoglutarate in an irreversible manner with no release of the transferred ammonia group into the medium. Identical results were obtained using thio-NADPH and acetylpyridine-NADPH, which are shown to be less efficient substrates of the enzyme than NADPH. Similarly, no exchange of the ammonia group being transferred with exogenous ammonium ion was observed during catalysis. The glutamate formed as the product of the iminoglutarate reduction was determined to be in the L configuration. The enzyme was also found to catalyze, under anaerobic conditions, the exchange of the 4proS H of NADPH with solvent both in the absence and in the presence of 2-oxoglutarate and glutamine. The reductive half-reaction is therefore a reversible segment of the overall irreversible amidotransferase reaction. 15N NMR studies also showed that the enzyme does not catalyze glutamate dehydrogenase/oxidase reactions or any observable glutaminase activity under neutral (pH 7.5) conditions. Glutaminase activity was also not observable with the reduced enzyme alone or in the presence of D-glutamate (a competitive inhibitor of glutamate synthase with respect to 2-oxoglutarate, with a Ki of about 11 microM) or with the oxidized enzyme in the presence of 2-oxoglutarate, D-glutamate, or NADP+. These data confirm species-dependent differences of A. brasilense glutamate synthase with respect to the enzyme from other sources.     

Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase

Glucosamine-6P synthase catalyzes the synthesis of glucosamine-6P from fructose-6P and glutamine and uses a channel to transfer ammonia from its glutaminase to its synthase active site. X-ray structures of glucosamine-6P synthase have been determined at 2.05 Angstroms resolution in the presence of fructose-6P and at 2.35 Angstroms resolution in the presence of fructose-6P and 6-diazo-5-oxo-L-norleucine, a glutamine affinity analog that covalently modifies the N-terminal catalytic cysteine, therefore mimicking the gamma-glutamyl-thioester intermediate formed during hydrolysis of glutamine. The fixation of the glutamine analog activates the enzyme through several major structural changes: 1) the closure of a loop to shield the glutaminase site accompanied by significant domain hinging, 2) the activation of catalytic residues involved in glutamine hydrolysis, i.e. the alpha-amino group of Cys-1 and Asn-98 that is positioned to form the oxyanion hole, and 3) a 75 degrees rotation of the Trp-74 indole group that opens the ammonia channel.     

Reaction coupling through interdomain contacts in imidazole glycerol phosphate synthase

Imidazole glycerol phosphate (IGP) synthase, a triad glutamine amidotransferase, catalyzes the fifth step in the histidine biosynthetic pathway, where ammonia from glutamine is incorporated into N1-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to yield IGP and 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR). The triad family of glutamine amidotransferases is formed by the coupling of two disparate subdomains, an acceptor domain and a glutamine hydrolysis domain. Each of the enzymes in this family share a common glutaminase domain for which the glutaminase activity is tightly regulated by an acceptor substrate domain. In IGP synthase the glutaminase and PRFAR binding sites are separated by 30 A. Using kinetic analyses of site-specific mutants and molecular dynamic simulations, we have determined that an interdomain salt bridge in IGP synthase between D359 and K196 (approximately 16 A from the PRFAR binding site) plays a key role in mediating communication between the two active sites. This interdomain contact modulates the glutaminase loop containing the histidine and glutamic acid of the catalytic triad to control glutamine hydrolysis. We propose this to be a general principle of catalytic coupling that may be applied to the entire triad glutamine amidotransferase family.     

Substrate-induced changes in the ammonia channel for imidazole glycerol phosphate synthase

IGP synthase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the unusual nucleotide, N(1)-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to form 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR) and imidazole glycerol phosphate (IGP). A common feature of all glutamine amidotransferases is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate. A refined assay system was developed to establish that Saccharomyces cerevisae IGP synthase shows a 4900-fold stimulation of glutaminase in the presence of the substrate acceptor PRFAR. The structure and function of IGP synthase acceptor substrate binding site were probed with competitive inhibitors that are nucleotide substrate and product analogues. In addition, these analogues were also used to establish that the normal steady-state turnover cycle involves a random sequential mechanism. Upregulation of the glutaminase active site occurs when these competitive inhibitors bind in the nucleotide site over 30 A away. One of the key structural features of IGP synthase is that the transfer of ammonia from the glutaminase site occurs through the (beta/alpha)(8) core of the protein. Upon the basis of the recent substrate-occupied structure for yeast IGP synthase (1), kinetic investigations of site-directed mutants revealed that a conserved K258 residue is key to productive binding and the overall stoichiometry of the reaction. The binding of the ribulosyl phosphate portion of the substrate PRFAR appears to be transduced through reorientation of K258 resulting in a conformational switch at the base of the (beta/alpha)(8) core that enables the passage of ammonia through the core of the protein. The overall analysis also leads to further discussion of how the residues that cover the opening of the (beta/alpha)(8) in the closed state may assist the channeling of ammonia at the interface of the two functional domains in the open state.     

Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.     

Dynamics of glucosamine-6-phosphate synthase catalysis

Glucosamine-6P synthase, which catalyzes glucosamine-6P synthesis from fructose-6P and glutamine, channels ammonia over 18 Å between its glutaminase and synthase active sites. The crystal structures of the full-length Escherichia coli enzyme have been determined alone, in complex with the first bound substrate, fructose-6P, in the presence of fructose-6P and a glutamine analog, and in the presence of the glucosamine-6P product. These structures represent snapshots of reaction intermediates, and their comparison sheds light on the dynamics of catalysis. Upon fructose-6P binding, the C-terminal loop and the glutaminase domains get ordered, leading to the closure of the synthase site, the opening of the sugar ring and the formation of a “closed” ammonia channel. Then, glutamine binding leads to the closure of the Q-loop to protect the glutaminase site, the activation of the catalytic residues involved in glutamine hydrolysis, the swing of the side chain of Trp74, which allows the communication between the two active sites through an “open” channel, and the rotation of the glutaminase domains relative to the synthase domains dimer. Therefore, binding of the substrates at the appropriate reaction time is responsible for the formation and opening of the ammonia channel and for the activation of the enzyme glutaminase function.     

Domain motions of glucosamine-6P synthase: comparison of the anisotropic displacements in the crystals and the catalytic hinge-bending rotation

Glucosamine-6-phosphate synthase channels ammonia over 18 A from glutamine at the glutaminase site to fructose-6P at the synthase site. We have modeled the anisotropic displacements of the glutaminase and synthase domains from the two crystallized states, the enzyme in complex with fructose-6P or in complex with glucose-6P and a glutamine affinity analog, using TLS (rigid-body motion in terms of translation, libration, and screw motions) refinement implemented in REFMAC. The domains displacements in the crystal lattices are compared to the movement of the glutaminase domain relative to the synthase domain that occurs during the catalytic cycle upon glutamine binding, which was visualized by comparing the two structures. This movement was analyzed by the program DYNDOM as a 22.8 degrees rotation around an effective hinge axis running approximately parallel to helix 300-317 of the synthase domain, the glutaminase loop that covers the glutaminase site upon glutamine binding acting as the mechanical hinge.     

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.     

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.     

Alpha-adrenergic stimulation of glutamine metabolism in isolated rat hepatocytes.

The mechanisms by means of which phenylephrine stimulates glutamine metabolism were studied in isolated rat hepatocytes. In the first 2 min after phenylephrine addition there was a rapid fall in the concentrations of intracellular 2-oxoglutarate and glutamate, presumably owing to activation of 2-oxoglutarate dehydrogenase. This was followed 2-3 min later by activation of glutaminase and by increases in glutamate and 2-oxoglutarate. Activation of glutaminase by phenylephrine was due to direct stimulation of the enzyme rather than to reversal of inhibition by the decrease in 2-oxoglutarate and glutamate. The stimulation of glutaminase by phenylephrine is partly due to an increase in the affinity of the enzyme for ammonia, its essential activator. It is concluded that stimulation of steady-state flux through the pathway from glutamine to glucose and urea can only be achieved by stimulation of glutaminase, the first enzyme in the pathway.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

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.     

Cross-talk and ammonia channeling between active centers in the unexpected domain arrangement of glutamate synthase

INTRODUCTION: The complex iron-sulfur flavoprotein glutamate synthase catalyses the reductive synthesis of L-glutamate from 2-oxoglutarate and L-glutamine, a reaction in the plant and bacterial pathway for ammonia assimilation. The enzyme functions through three distinct active centers carrying out L-glutamine hydrolysis, conversion of 2-oxoglutarate into L-glutamate, and electron uptake from an electron donor. RESULTS: The 3.0 A crystal structure of the dimeric 324 kDa core protein of a bacterial glutamate synthase was solved by the MAD method, using the very weak anomalous signal of the two 3Fe-4S clusters present in the asymmetric unit. The 1,472 amino acids of the monomer fold into a four-domain architecture. The two catalytic domains have canonical Ntn-amidotransferase and FMN binding (beta/alpha)8 barrel folds, respectively. The other two domains have an unusual "cut (beta/alpha)8 barrel" topology and an unexpected novel beta-helix structure. Channeling of the ammonia intermediate is brought about by an internal tunnel of 31 A length, which runs from the site of L-glutamine hydrolysis to the site of L-glutamate synthesis. CONCLUSIONS: The outstanding property of glutamate synthase is the ability to coordinate the activity of its various functional sites to avoid wasteful consumption of L-glutamine. The structure reveals two polypeptide segments that connect the catalytic centers and embed the ammonia tunnel, thus being ideally suited to function in interdomain signaling. Depending on the enzyme redox and ligation states, these signal-transducing elements may affect the active site geometry and control ammonia diffusion through a gating mechanism.     

Inhibition of E. coli CTP synthase by the "positive" allosteric effector GTP

Cytidine 5'-triphosphate (CTP) synthase catalyzes the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as a positive allosteric effector to promote catalysis of glutamine hydrolysis. We show that at concentrations exceeding approximately 0.15 mM, GTP actually behaves as a negative allosteric effector of E. coli CTP synthase, inhibiting glutamine-dependent CTP formation. In addition, GTP inhibits NH(3)-dependent CTP formation in a concentration-dependent manner. However, GTP does not inhibit the enzyme's intrinsic glutaminase activity. Although the activation of CTP synthase by GTP does not display cooperative behavior, inhibition of both CTP synthase-catalyzed ammonia- and glutamine-dependent CTP synthesis by GTP do exhibit positive cooperativity. These results suggest that GTP binding affects CTP synthase catalysis in two ways: it activates enzyme-catalyzed glutamine hydrolysis and it inhibits the utilization of NH(3) as a substrate by the synthase domain.     

Toward understanding the mechanism of the complex cyclization reaction catalyzed by imidazole glycerolphosphate synthase: crystal structures of a ternary complex and the free enzyme

Imidazole glycerol phosphate synthase catalyzes formation of the imidazole ring in histidine biosynthesis. The enzyme is also a glutamine amidotransferase, which produces ammonia in a glutaminase active site and channels it through a 30-A internal tunnel to a cyclase active site. Glutaminase activity is impaired in the resting enzyme, and stimulated by substrate binding in the cyclase active site. The signaling mechanism was investigated in the crystal structure of a ternary complex in which the glutaminase active site was inactivated by a glutamine analogue and the unstable cyclase substrate was cryo-trapped in the active site. The orientation of N(1)-(5'-phosphoribulosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide in the cyclase active site implicates one side of the cyclase domain in signaling to the glutaminase domain. This side of the cyclase domain contains the interdomain hinge. Two interdomain hydrogen bonds, which do not exist in more open forms of the enzyme, are proposed as molecular signals. One hydrogen bond connects the cyclase domain to the substrate analogue in the glutaminase active site. The second hydrogen bond connects to a peptide that forms an oxyanion hole for stabilization of transient negative charge during glutamine hydrolysis. Peptide rearrangement induced by a fully closed domain interface is proposed to activate the glutaminase by unblocking the oxyanion hole. This interpretation is consistent with biochemical results [Myers, R. S., et al., (2003) Biochemistry 42, 7013-7022, the accompanying paper in this issue] and with structures of the free enzyme and a binary complex with a second glutamine analogue.     

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.     

The pathway of nitrogen assimilation in plants

The major route of nitrogen assimilation has been considered for many years to occur via the reductive amination of α-oxoglutarate, catalysed by glutamate dehydrogenase. However, recent work has shown that in most bacteria an alternative route via glutamine synthetase and glutamine: 2-oxoglutarate aminotransferase (glutamate synthase) operates under conditions of ammonia limitation. Subsequently the presence of a ferredoxin-dependent glutamate synthase in green leaves and green and blue-green algae, and a NAD(P)H and ferredoxin-dependent enzyme in roots and other non-green plant tissues, has suggested that this route may also function in most members of the plant kingdom. The only exceptions are probably the majority of the fungi, where so far most organisms studied do not appear to contain glutamate synthase. Besides the presence of the necessary enzymes there is other evidence to support the contention that the assimilation of ammonia into amino acids occurs via glutamine synthetase and glutamate synthase, and that it is unlikely that glutamate dehydrogenase plays a major role in nitrogen assimilation in bacteria or higher plants except in circumstances of ammonia excess.     

Transport of glutamine by Streptococcus bovis and conversion of glutamine to pyroglutamic acid and ammonia

Streptococcus bovis JB1 cells energized with glucose transported glutamine at a rate of 7 nmol/mg of protein per min at a pH of 5.0 to 7.5; sodium had little effect on the transport rate. Because valinomycin-treated cells loaded with K and diluted into Na (pH 6.5) to create an artificial delta psi took up little glutamine, it appeared that transport was driven by phosphate-bond energy rather than proton motive force. The kinetics of glutamine transport by glucose-energized cells were biphasic, and it appeared that facilitated diffusion was also involved, particularly at high glutamine concentrations. Glucose-depleted cultures took up glutamine and produced ammonia, but the rate of transport per unit of glutamine (V/S) by nonenergized cells was at least 1,000-fold less than the V/S by glucose-energized cells. Glutamine was converted to pyroglutamate and ammonia by a pathway that did not involve a glutaminase reaction or glutamate production. No ammonia production from pyroglutamate was detected. S. bovis was unable to take up glutamate, but intracellular glutamate concentrations were as high as 7 mM. Glutamate was produced from ammonia via a glutamate dehydrogenase reaction. Cells contained high concentrations of 2-oxoglutarate and NADPH that inhibited glutamate deamination and favored glutamate formation. Since the carbon skeleton of glutamine was lost as pyroglutamate, glutamate formation occurred at the expense of glucose. Arginine deamination is often used as a taxonomic tool in classifying streptococci, and it had generally been assumed that other amino acids could not be fermented. To our knowledge, this is the first report of glutamine conversion to pyroglutamate and ammonia in streptococci.     

Imidazole glycerol phosphate synthase from Thermotoga maritima. Quaternary structure, steady-state kinetics, and reaction mechanism of the bienzyme complex

Imidazole glycerol phosphate synthase, which links histidine and de novo purine biosynthesis, is a member of the glutamine amidotransferase family. In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme complex of the glutaminase subunit HisH and the synthase subunit HisF. Nascent ammonia produced by HisH reacts at the active site of HisF with N'-((5'-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide. In order to elucidate the interactions between HisH and HisF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coli, purified, and characterized. Isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF to which either the product imidazole glycerol phosphate or a substrate analogue were bound. Eight conserved amino acids at the putative active site of tHisF were exchanged by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics. Aspartate 11 appeared to be essential for the synthase activity both in vitro and in vivo, and aspartate 130 could be partially replaced only by glutamate. The carboxylate groups of these residues could provide general acid/base catalysis in the proposed catalytic mechanism of the synthase reaction.