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.     

Expression and purification of imidazole glycerol phosphate synthase from Saccharomyces cerevisiae

Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase that catalyzes the formation of IGP and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) from N(1)-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-car boxamide ribonucleotide (PRFAR). This enzyme represents a junction between histidine biosynthesis and de novo purine biosynthesis. The recent characterization of the HIS7 gene in the yeast Saccharomyces cerevisiae IGP synthase established that this protein is bifunctional, representing a fusion between the N-terminal HisH domain and a C-terminal HisF domain. Catalytically active yeast HIS7 was expressed in a bacterial system under the control of T7 polymerase promoter. The recombinant enzyme was purified to homogeneity and the native molecular weight and steady-state kinetic constants were determined. The yeast enzyme is distinguished from the Escherichia coli IGP synthase in its utilization of ammonia as a substrate. HIS7 displays a higher K(m) for glutamine and a lower turnover in the ammonia-dependent IGP synthase activity. As observed with the E. coli IGP synthase, HIS7 shows a low basal level glutaminase activity that can be enhanced 1000-fold in the presence of a nucleotide substrate or analog. The purification and characterization of the S. cerevisiae enzyme will enable a more detailed investigation of the biochemical mechanisms that mediate the ammonia-transfer process. The fused structural feature of the HIS7 protein and the development of a high-level production system for the active enzyme elevate the potential for determination of its three-dimensional structure through X-ray crystallography.     

Expression and Purification of Imidazole Glycerol Phosphate Synthase from Saccharomyces cerevisiae

Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase that catalyzes the formation of IGP and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) from N¹-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR). This enzyme represents a junction between histidine biosynthesis and de novo purine biosynthesis. The recent characterization of the HIS7 gene in the yeast Saccharomyces cerevisiae IGP synthase established that this protein is bifunctional, representing a fusion between the N-terminal HisH domain and a C-terminal HisF domain. Catalytically active yeast HIS7 was expressed in a bacterial system under the control of T7 polymerase promoter. The recombinant enzyme was purified to homogeneity and the native molecular weight and steady-state kinetic constants were determined. The yeast enzyme is distinguished from the Escherichia coli IGP synthase in its utilization of ammonia as a substrate. HIS7 displays a higher K_m for glutamine and a lower turnover in the ammonia-dependent IGP synthase activity. As observed with the E. coli IGP synthase, HIS7 shows a low basal level glutaminase activity that can be enhanced 1000-fold in the presence of a nucleotide substrate or analog. The purification and characterization of the S. cerevisiae enzyme will enable a more detailed investigation of the biochemical mechanisms that mediate the ammonia-transfer process. The fused structural feature of the HIS7 protein and the development of a high-level production system for the active enzyme elevate the potential for determination of its three-dimensional structure through X-ray crystallography.     

Structural elements in IGP synthase exclude water to optimize ammonia transfer

In the complex pathway of histidine biosynthesis, a key branch point linking amino acid and purine biosynthesis is catalyzed by the bifunctional enzyme imidazole glycerol phosphate (IGP) synthase. The first domain of IGP synthase, a triad glutamine amidotransferase, hydrolyzes glutamine to form glutamate and ammonia. Its activity is tightly regulated by the binding of the substrate PRFAR to its partner synthase domain. Recent crystal structures and molecular dynamics simulations strongly suggest that the synthase domain, a (beta/alpha)(8) barrel protein, mediates the insertion of ammonia and ring formation in IGP by channeling ammonia from one remote active site to the other. Here, we combine both mutagenesis experiments and computational investigations to gain insight into the transfer of ammonia and the mechanism of conduction. We discover an alternate route for the entrance of ammonia into the (beta/alpha)(8) barrel and argue that water acts as both agonist and antagonist to the enzymatic function. Our results indicate that the architecture of the two subdomains, most notably the strict conservation of key residues at the interface and within the (beta/alpha)(8) barrel, has been optimized to allow the efficient passage of ammonia, and not water, between the two remote active sites.     

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.     

Millisecond dynamics in the allosteric enzyme imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima

IGPS is a 51 kDa heterodimeric enzyme comprised of two proteins, HisH and HisF, that catalyze the hydrolysis of glutamine to produce NH₃ in the HisH active site and the cyclization of ammonia with N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in HisF to produce imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR). Binding of PRFAR and IGP stimulates glutaminase activity in the HisH enzyme over 5,000 and 100-fold, respectively, despite the active sites being >25 Å apart. The details of this long-range protein communication process were investigated by solution NMR spectroscopy and CPMG relaxation dispersion experiments. Formation of the heterodimer enzyme results in a reduction in millisecond motions in HisF that extend throughout the protein. Binding of lGP results in an increase in protein-wide millisecond dynamics evidenced as severe NMR line broadening and elevated R _ex values. Together, these data demonstrate a grouping of flexible residues that link the HisF active site with the protein interface to which HisH binds and provide a model for the path of communication between the IGPS active sites.     

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.     

A network of conserved interactions regulates the allosteric signal in a glutamine amidotransferase

We have combined equilibrium and steered molecular dynamics (SMD) simulations with principal component and correlation analyses to probe the mechanism of allosteric regulation in imidazole glycerol phosphate (IGP) synthase. An evolutionary analysis of IGP synthase revealed a conserved network of interactions leading from the effector binding site to the glutaminase active site, forming conserved communication pathways between the remote active sites. SMD simulations of the undocking of the ribonucleotide effector N1-[(5'-phosphoribulosyl)-formino]-5'-aminoimidazole carboxamide ribonucleotide (PRFAR) resulted in a large scale hinge-opening motion at the interface. Principal component analysis and a correlation analysis of the equilibration protein motion indicate that the dynamics involved in the allosteric transition are mediated by coupled motion between sites that are more than 25 A apart. Furthermore, conserved residues at the substrate-binding site, within the barrel, and at the interface were found to exhibit highly correlated motion during the allosteric transition. The coupled motion between PRFAR unbinding and the directed opening of the interface is interpreted in combination with kinetic assays for the wild-type and mutant systems to develop a model of allosteric regulation in IGP synthase that is monitored and investigated with atomic resolution.     

Crystal structure of imidazole glycerol phosphate synthase: a tunnel through a (beta/alpha)8 barrel joins two active sites

BACKGROUND: Imidazole glycerol phosphate synthase catalyzes a two-step reaction of histidine biosynthesis at the bifurcation point with the purine de novo pathway. The enzyme is a new example of intermediate channeling by glutamine amidotransferases in which ammonia generated by hydrolysis of glutamine is channeled to a second active site where it acts as a nucleophile. In this case, ammonia reacts in a cyclase domain to produce imidazole glycerol phosphate and an intermediate of purine biosynthesis. The enzyme is also a potential target for drug and herbicide development since the histidine pathway does not occur in mammals. RESULTS: The 2.1 A crystal structure of imidazole glycerol phosphate synthase from yeast reveals extensive interaction of the glutaminase and cyclase catalytic domains. At the domain interface, the glutaminase active site points into the bottom of the (beta/alpha)(8) barrel of the cyclase domain. An ammonia tunnel through the (beta/alpha)(8) barrel connects the glutaminase docking site at the bottom to the cyclase active site at the top. A conserved "gate" of four charged residues controls access to the tunnel. CONCLUSIONS: This is the first structure in which all the components of the ubiquitous (beta/alpha)(8) barrel fold, top, bottom, and interior, take part in enzymatic function. Intimate contacts between the barrel domain and the glutaminase active site appear to be poised for crosstalk between catalytic centers in response to substrate binding at the cyclase active site. The structure provides a number of potential sites for inhibitor development in the active sites and in a conserved interdomain cavity.     

Structural evidence for ammonia tunneling across the (beta alpha)(8) barrel of the imidazole glycerol phosphate synthase bienzyme complex

Since reactive ammonia is not available under physiological conditions, glutamine is used as a source for the incorporation of nitrogen in a number of metabolic pathway intermediates. The heterodimeric ImGP synthase that links histidine and purine biosynthesis belongs to the family of glutamine amidotransferases in which the glutaminase activity is coupled with a subsequent synthase activity specific for each member of the enzyme family. Its X-ray structure from the hyperthermophile Thermotoga maritima shows that the glutaminase subunit is associated with the N-terminal face of the (beta alpha)(8) barrel cyclase subunit. The complex reveals a putative tunnel for the transfer of ammonia over a distance of 25 A. Although ammonia tunneling has been reported for glutamine amidotransferases, the ImGP synthase has evolved a novel mechanism, which extends the known functional properties of the versatile (beta alpha)(8) barrel fold.     

Conformational changes in ammonia-channeling glutamine amidotransferases

Glutamine amidotransferases (GATs), which catalyze the synthesis of different aminated products, channel ammonia over 10-40 A from a glutamine substrate at the glutaminase site to an acceptor substrate at the synthase site. Ammonia production usually uses a cysteine-histidine-glutamate triad or a N-terminal cysteine residue. Crystal structures of several amidotransferase ligand complexes, mimicking intermediates along the catalytic cycle, have now been determined. In most cases, acceptor binding triggers glutaminase activation through domain-hinged movements and other conformational changes. Structural information shows how flexible loops of the synthase and glutaminase domains move to shield the two catalytic sites and anchor the substrates, and how the ammonia channel forms and opens or closes.     

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.     

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.     

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.     

Molecular evolution of the histidine biosynthetic pathway

The available sequences of genes encoding the enzymes associated with histidine biosynthesis suggest that this is an ancient metabolic pathway that was assembled prior to the diversification of the Bacteria, Archaea, and Eucarya. Paralogous duplications, gene elongation, and fusion events involving different his genes have played a major role in shaping this biosynthetic route. Evidence that the hisA and the hisF genes and their homologues are the result of two successive duplication events that apparently took place before the separation of the three cellular lineages is extended. These two successive gene duplication events as well as the homology between the hisH genes and the sequences encoding the TrpG-type amidotransferases support the idea that during the early stages of metabolic evolution at least parts of the histidine biosynthetic pathway were mediated by enzymes of broader substrate specificities. Maximum likelihood trees calculated for the available sequences of genes encoding these enzymes have been obtained. Their topologies support the possibility of an evolutionary proximity of archaebacteria with low GC Gram-positive bacteria. This observation is consistent with those detected by other workers using the sequences of heat-shock proteins (HSP70), glutamine synthetases, glutamate dehydrogenases, and carbamoylphosphate synthetases.Abbreviations as amino acid - ORF open reading frame - bp base pair - kb 10³ bp - CarA carbamoyl phosphate synthetase (EC 6.3.5.5) - GAT glutamine amidotransferase - GuaA GMP synthetase (EC 6.3.4.1) - PabA 4-amino-4-deoxychorismate synthase (EC 4.1.3-) - PyrG GTP synthetase (EC 6.3.4.2) - AICAR 5-aminoimidazole-4-carboxamide-l--d ribofuranosyl 5-monophosphate - HAL l-histidinal - HOL l-histidinol - HP histidinol phosphate - IAP imidazole acetol-phosphate - IGP imidazole glycerol phosphate - PR phosphoribosyl - PRFAR N-[(5-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide - 5-ProFAR N ¹-[(5-phosphoribosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide - PRPP phosphoribosyl-pyrophosphate - RFLP restriction fragment length polymorphism Correspondence to: R. Fani     

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.     

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.     

Structure--function studies on the iron-sulfur flavoenzyme glutamate synthase: an unexpectedly complex self-regulated enzyme

Glutamate synthase (GltS) is, with glutamine synthetase, the key enzyme of ammonia assimilation in bacteria, microorganisms and plants. GltS isoforms result from the assembly and co-evolution of conserved functional domains. They share a common mechanism of reductive glutamine-dependent glutamate synthesis from 2-oxoglutarate, which takes place within the alpha subunit ( approximately 150 kDa) of the NADPH-dependent bacterial enzyme and the corresponding polypeptides of other GltS forms, and involves: (i) an Ntn-type amidotransferase domain and (ii) a flavin mononucleotide-containing (beta/alpha)(8) barrel synthase domain connected by (iii) a approximately 30 A-long intramolecular ammonia tunnel. The synthase domain harbors the [3Fe/4S](0,+1) cluster of the enzyme, which participates in the electron transfer process from the physiological reductant: reduced ferredoxin in the plant-type enzyme or NAD(P)H in the bacterial and the non-photosynthetic eukaryotic form. The NAD(P)H-dependent GltS requires a tightly bound flavin adenine dinucleotide-dependent reductase (beta subunit, approximately 50 kDa), also determining the presence of two low-potential [4Fe-4S](+1,+2) clusters. Structural, functional and computational data available on GltS and related enzymes show how the enzyme may control and coordinate the reactions taking place at the glutaminase and synthase sites by sensing substrate binding and cofactor redox state.     

Structural insights into the mechanism of the PLP synthase holoenzyme from Thermotoga maritima

Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6 and is an important cofactor for several of the enzymes involved in the metabolism of amine-containing natural products such as amino acids and amino sugars. The PLP synthase holoenzyme consists of two subunits: YaaD catalyzes the condensation of ribulose 5-phosphate, glyceraldehyde-3-phosphate, and ammonia, and YaaE catalyzes the production of ammonia from glutamine. Here we describe the structure of the PLP synthase complex (YaaD-YaaE) from Thermotoga maritima at 2.9 A resolution. This complex consists of a core of 12 YaaD monomers with 12 noninteracting YaaE monomers attached to the core. Compared with the previously published structure of PdxS (a YaaD ortholog in Geobacillus stearothermophilus), the N-terminus (1-18), which includes helix alpha0, the beta2-alpha2 loop (46-56), which includes new helix alpha2a, and the C-terminus (270-280) of YaaD are ordered in the complex but disordered in PdxS. A ribulose 5-phosphate is bound to YaaD via an imine with Lys82. Previous studies have demonstrated a similar imine at Lys149 and not at Lys81 (equivalent to Lys150 and Lys82 in T. maritima) for the Bacillus subtilis enzyme suggesting the possibility that two separate sites on YaaD are involved in PLP formation. A phosphate from the crystallization solution is found bound to YaaD and also serves as a marker for a possible second active site. An ammonia channel that connects the active site of YaaE with the ribulose 5-phosphate binding site was identified. This channel is similar to one found in imidazole glycerol phosphate synthase; however, when the beta-barrels of the two complexes are superimposed, the glutaminase domains are rotated by about 180 degrees with respect to each other.