Intramolecular Tunnel Research Articles

3D Variability Analysis Reveals a Hidden Conformational Change Controlling Ammonia Transport in Human Asparagine Synthetase.

How motions in enzymes might be linked to catalytic function is of considerable general interest. Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we use 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) to identify a functional role for the Arg-142 side chain as a gate that mediates ammonia access to a catalytically relevant intramolecular tunnel. Our 3DVA-derived hypothesis is assessed experimentally, using the R142I variant in which Arg-142 is replaced by isoleucine, and by molecular dynamics (MD) simulations on independent, computational models of the WT human ASNS monomer and its catalytically relevant, ternary complex with β-aspartyl-AMP and MgPPi. Residue fluctuations in the MD trajectories for the human ASNS monomer are consistent with those determined for 3DVA-derived structures. These MD simulations also indicate that the gating function of Arg-142 is separate from the molecular events that form a continuous tunnel linking the two active sites. Experimental support for Arg-142 playing a role in intramolecular ammonia translocation is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS. MD simulations of computational models for the R142I variant and the R142I/β-aspartyl-AMP/MgPPi ternary complex provide a possible molecular basis for this observation. Overall, the combination of 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.

Heterologous expression, purification, and characterization of a recombinant Cordyceps militaris lipase from Candida rugosa-like family in Pichia pastoris

Multiple sequence alignments of three lipase isoforms from the filamentous fungus, Cordyceps militaris, have revealed that the deduced protein from their common sequence belongs to the Candida rugosa lipase-like group. To express the protein in its active form, recombinant lipase from C. militaris (rCML) was extra cellularly expressed in Pichia pastoris X-33 after removing its signal peptide. Purified rCML was a stable monomeric protein with a molecular mass of 90 kDa, and was highly N-mannosylated compared to the native protein (69 kDa). The catalytic efficiency (kcat/Km) of rCML was greater than the native protein (1244.35 ± 50.88 and 1067.17 ± 29.07 mM−1·min−1, respectively), yet they had similar optimal pH values and temperatures (40 °C and pH 7.0–7.5), and showed preferences for Tween esters and short-chain triacylglycerols. Despite its monomeric conformation, interfacial activation was not observed for rCML, unlike the classical lipases. From the structural model of rCML, the binding pocket of rCML was predicted as a funnel-like structure consisting of a hollow space and an intramolecular tunnel, which is typical of C. rugosa lipase-like lipases. However, a blockage shortened the tunnel to 12–15 Å, which endows strict short-chain selectivity towards triacylglycerols and a perfect match for tricaproin (C6:0). The limited depth of the tunnel may enable accommodation of triacylglycerols with medium-to-long-chain fatty acids, which differentiates rCML from other C. rugosa lipase-like lipases with broad substrate specificities.

GTP-Dependent Regulation of CTP Synthase: Evolving Insights into Allosteric Activation and NH3 Translocation.

Cytidine-5′-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of Drosophila CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible “wing” region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH3 tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.

Opposite Charge Movements Within the Photoactive Site Modulate Two-Step Channel Closing in GtACR1

Guillardia theta anion channelrhodopsin 1 is a light-gated anion channel widely used as an optogenetic inhibitory tool. Our recently published crystal structure of its dark (closed) state revealed that the photoactive retinylidene chromophore is located midmembrane in a full-length intramolecular tunnel through the protein, the radius of which is less than that of a chloride ion. Here we show that acidic (glutamate) substitutions for residues within the inner half-tunnel enhance the fast channel closing and, for residues within the outer half-tunnel, enhance the slow channel closing. The magnitude of these effects was proportional to the distance of the mutated residue from the photoactive site. These data indicate that the local electrical field across the photoactive site controls fast and slow channel closing, involving outward and inward charge displacements. In the purified mutant proteins, we observed corresponding opposite changes in kinetics of the M photocycle intermediate. A correlation between fast closing and M rise and slow closing and M decay observed in the mutants suggests that the Schiff base proton is one of the displaced charges. Opposite signs of the effects indicate that deprotonation and reprotonation of the Schiff base take place on the same (outer) side of the membrane and explains opposite rectification of fast and slow channel closing. Оur comprehensive protein-wide acidic residue substitution screen shows that only mutations of the residues located in the intramolecular tunnel confer strong rectification, which confirms the prediction that the tunnel expands upon photoexcitation to form the anion pathway.

Design, synthesis, and evaluation of substituted nicotinamide adenine dinucleotide (NAD+) synthetase inhibitors as potential antitubercular agents

Nicotinamide adenine dinucleotide (NAD+) synthetase catalyzes the last step in NAD+ biosynthesis. Depletion of NAD+ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD+ synthetase (NadE) from Mtb, we expect to eliminate NAD+ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure–activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC50=1000µM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC50 value of 90µM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19–100µg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.

Evolutionary history of versatile-lipases from Agaricales through reconstruction of ancestral structures

BackgroundFungal “Versatile carboxylic ester hydrolases” are enzymes with great biotechnological interest. Here we carried out a bioinformatic screening to find these proteins in genomes from Agaricales, by means of searching for conserved motifs, sequence and phylogenetic analysis, and three-dimensional modeling. Moreover, we reconstructed the molecular evolution of these enzymes along the time by inferring and analyzing the sequence of ancestral intermediate forms.ResultsThe properties of the ancestral candidates are discussed on the basis of their three-dimensional structural models, the hydrophobicity of the lid, and the substrate binding intramolecular tunnel, revealing all of them featured properties of these enzymes. The evolutionary history of the putative lipases revealed an increase on the length and hydrophobicity of the lid region, as well as in the size of the substrate binding pocket, during evolution time. These facts suggest the enzymes’ specialization towards certain substrates and their subsequent loss of promiscuity.ConclusionsThese results bring to light the presence of different pools of lipases in fungi with different habitats and life styles. Despite the consistency of the data gathered from reconstruction of ancestral sequences, the heterologous expression of some of these candidates would be essential to corroborate enzymes’ activities.

Computational investigation of O2 diffusion through an intra-molecular tunnel in AlkB; influence of polarization on O2 transport.

E. Coli AlkB catalyzes the direct dealkylation of various alkylated bases in damaged DNA. The diffusion of molecular oxygen to the active site in AlkB is an essential step for the oxidative dealkylation activity. Despite detailed studies on the stepwise oxidation mechanism of AlkB, there is no conclusive picture of how O2 molecules reach the active site of the protein. Yu et al. (Nature, 439, 879) proposed the existence of an intra-molecular tunnel based on their initial crystal structures of AlkB. We have employed computational simulations to investigate possible migration pathways inside AlkB for O2 molecules. Extensive molecular dynamics (MD) simulations, including explicit ligand sampling and potential of mean force (PMF) calculations, have been performed to provide a microscopic description of the O2 delivery pathway in AlkB. Analysis of intra-molecular tunnels using the CAVER software indicates two possible pathways for O2 to diffuse into the AlkB active site. Explicit ligand sampling simulations suggests that only one of these tunnels provides a viable route. The free energy path for an oxygen molecule to travel along each of these tunnels has been determined with AMBER and AMOEBA. Both PMFs indicate passive transport of O2 from the surface of the protein. However, the inclusion of explicit polarization shows a very large barrier for diffusion of the co-substrate out of the active site, compared with the non-polarizable potential. In addition, our results suggest that the mutation of a conserved residue along the tunnel, Y178, has dramatic effects on the dynamics of AlkB and on the transport of O2 along the tunnel.

The Impact of Perturbing Dynamic Amino Acid Networks in a (β/α)8 Barrel Enzyme

Amino acid networks describe the web of noncovalent interactions between residues spanning an enzyme. These networks may be responsible for the propagation of regulatory signals across the protein that influence conformation, binding of substrate(s), and catalysis. Tryptophan synthase (TS), the final enzyme in the tryptophan biosynthetic pathway, is a tetramer consisting of a pair of alpha and beta heterodimers arranged in a linear conformation. The alpha and beta subunits are connected by a 25Å intramolecular tunnel that channels indole, a product from the alpha reaction, to the active site in the beta subunit. In addition to this tunnel, the conformational states of these subunits are highly coordinated making TS an ideal and heavily studied model for substrate channeling and enzyme-enzyme interactions. We used nuclear magnetic resonance chemical shift covariance analysis to delineate amino acid networks in the alpha subunit, a (β/α)8 barrel enzyme. We have shown that these observed networks change between the resting state (in the absence of substrates) and the working state (under active catalytic turnover). Furthermore, the loss of a hydrogen bond between the dynamic β2α2 and β6α6 loops in the T183V variant significantly changes these networks and the catalytic rate seen in the wild-type. These networks were perturbed by making small Ala to Gly substitutions of surface residues correlated to the catalytic Glu49. These modifications resulted in modest decreases in the catalytic rate, although they are 25 Å away from the active site. Amino acid networks are important for the function of an enzyme and may be manipulated to tune its function.

The Tryptophan Synthase α2β2 Complex: A Model for Substrate Channeling, Allosteric Communication, and Pyridoxal Phosphate Catalysis

I reflect on my research on pyridoxal phosphate (PLP) enzymes over fifty-five years and on how I combined research with marriage and family. My Ph.D. research with Esmond E. Snell established one aspect of PLP enzyme mechanism. My postdoctoral work first with Hans L. Kornberg and then with Alton Meister characterized the structure and function of another PLP enzyme, l-aspartate β-decarboxylase. My independent research at the National Institutes of Health (NIH) since 1966 has focused on the bacterial tryptophan synthase α2β2 complex. The β subunit catalyzes a number of PLP-dependent reactions. We have characterized these reactions and the allosteric effects of the α subunit. We also used chemical modification to probe enzyme structure and function. Our crystallization of the tryptophan synthase α2β2 complex from Salmonella typhimurium led to the determination of the three-dimensional structure with Craig Hyde and David Davies at NIH in 1988. This landmark structure was the first structure of a multienzyme complex and the first structure revealing an intramolecular tunnel. The structure has provided a basis for exploring mechanisms of catalysis, channeling, and allosteric communication in the tryptophan synthase α2β2 complex. The structure serves as a model for many other multiprotein complexes that are important for biological processes in prokaryotes and eukaryotes.

A conserved glutamate controls the commitment to acyl-adenylate formation in asparagine synthetase.

Inhibitor docking studies have implicated a conserved glutamate residue (Glu-348) as a general base in the synthetase active site of the enzyme asparagine synthetase B from Escherichia coli (AS-B). We now report steady-state kinetic, isotope transfer, and positional isotope exchange experiments for a series of site-directed AS-B mutants in which Glu-348 is substituted by conservative amino acid replacements. We find that formation of the β-aspartyl-AMP intermediate, and therefore the eventual production of asparagine, is dependent on the presence of a carboxylate side chain at this position in the synthetase active site. In addition, Glu-348 may also play a role in mediating the conformational changes needed to (i) coordinate, albeit weakly, the glutaminase and synthetase activities of the enzyme and (ii) establish the structural integrity of the intramolecular tunnel along which ammonia is translocated. The importance of Glu-348 in mediating acyl-adenylate formation contrasts with the functional role of the cognate residues in β-lactam synthetase (BLS) and carbapenem synthetase (CPS) (Tyr-348 and Tyr-345, respectively), which both likely evolved from asparagine synthetase. Given the similarity of the chemistry catalyzed by AS-B, BLS, and CPS, our work highlights the difficulty of predicting the functional outcome of single site mutations on enzymes that catalyze almost identical chemical transformations.

Computational Studies of Ammonia Channel Function in Glutamine 5′-Phosphoribosylpyrophosphate Amidotransferase

Glutamine 5'-phosphoribosylpyrophosphate amidotransferase (GPATase) catalyzes the synthesis of 5'-phosphoribosylamine in a reaction that involves the translocation of ammonia along an intramolecular tunnel linking the two active sites of the enzyme. We now report a locally enhanced sampling (LES) strategy for modeling ammonia transfer between the active sites of Escherichia coli GPATase in its active conformation. Our calculations demonstrate that the ammonia channel in GPATase is best regarded as a "pipe" through which ammonia travels in the absence of an external "driving" potential. This combined LES/PMF computational approach, which offers a straightforward alternative to steered molecular dynamics simulations in studies of substrate channeling, also provides new insights into the molecular basis of the reduced ammonia transfer efficiency exhibited by the L415A GPATase mutant.

Mycolyltransferase from Mycobacterium leprae Excludes Mycolate-containing Glycolipid Substrates

Trehalose dimycolate (TDM) is a major surface-exposed mycolyl glycolipid that contributes to the hydrophobic cell wall architecture of mycobacteria. Nevertheless, because of its potent adjuvant functions, pathogenic mycobacteria appear to have evolved an evasive maneuver to down-regulate TDM expression within the host. We have shown previously that Mycobacterium tuberculosis (M.tb) and Mycobacterium avium (M.av), replace TDM with glucose monomycolate (GMM) by borrowing host-derived glucose as an alternative substrate for the FbpA mycolyltransferase. Mycobacterium leprae (M.le), the causative microorganism of human leprosy, is also known to down-regulate TDM expression in infected tissues, but the function of its mycolyltransferases has been poorly analysed. We found that, unlike M.tb and M.av FbpA enzymes, M.av FbpA was unexpectedly inefficient in transferring alpha-branched mycolates, resulting in impaired production of both TDM and GMM. Molecular modelling and mutational analysis indicated that a bulky side chain of leucine at position 130 of M.le FbpA obstructed the intramolecular tunnel that was proposed to accommodate the alpha-branch portion of the substrates. Notably, even after a highly reductive evolution, M.le FbpA remained functional in terms of transferring unbranched acyl chains, suggesting a role that is distinct from that as a mycolyltransferase.

Structure–function studies of glutamate synthases: A class of self‐regulated iron‐sulfur flavoenzymes essential for nitrogen assimilation

Glutamate synthases play with glutamine synthetase an essential role in nitrogen assimilation processes in microorganisms, plants, and lower animals by catalyzing the net synthesis of one molecule of L-glutamate from L-glutamine and 2-oxoglutarate. They exhibit a modular architecture with a common subunit or region, which is responsible for the L-glutamine-dependent glutamate synthesis from 2-oxoglutarate. Here, a PurF- (Type II- or Ntn-) type amidotransferase domain is coupled to the synthase domain, a (beta/alpha)8 barrel containing FMN and one [3Fe-4S]0,+1 cluster, through a approximately 30 angstroms-long intramolecular tunnel for the transfer of ammonia between the sites. In bacterial and eukaryotic GltS, reducing equivalents are provided by reduced pyridine nucleotides thanks to the stable association with a second subunit or region, which acts as a FAD-dependent NAD(P)H oxidoreductase and is responsible for the formation of the two low potential [4Fe-4S]+1,+2 clusters of the enzyme. In photosynthetic cells, reduced ferredoxin is the physiological reductant. This review focus on the mechanism of cross-activation of the synthase and glutaminase reactions in response to the bound substrates and the redox state of the enzyme cofactors, as well as on recent information on the structure of the alphabeta protomer of the NADPH-dependent enzyme, which sheds light on the intramolecular electron transfer pathway between the flavin cofactors.

Rectification effects in coherent transport through single molecules

A minimal model for coherent transport through a donor/acceptor molecular junction is presented. The two donor and acceptor sites are described by single levels energetically separated by an intramolecular tunnel barrier. In the limit of strong coupling to the electrodes a current rectification for different bias voltage polarities occurs. Contacts with recent experiments of molecular rectification are also given.

A Convenient gHMQC-Based NMR Assay for Investigating Ammonia Channeling in Glutamine-Dependent Amidotransferases: Studies of Escherichia coli Asparagine Synthetase B

X-ray crystal structures of glutamine-dependent amidotransferases in their "active" conformation have revealed the existence of multiple active sites linked by solvent inaccessible intramolecular channels, giving rise to the widely accepted view that ammonia released in a glutaminase site is channeled efficiently into a separate synthetase site where it undergoes further reaction. We now report a very convenient isotope-edited 1H NMR-based assay that can be used to probe the transfer of ammonia between the active sites of amidotransferases and demonstrate its use in studies of Escherichia coli asparagine synthetase B (AS-B). Our NMR results suggest that (i) high glutamine concentrations do not suppress ammonia-dependent asparagine formation in this bacterial asparagine synthetase and (ii) ammonia in bulk solution can react with the thioester intermediate formed during the glutaminase half-reaction by accessing the N-terminal active site of AS-B during catalytic turnover. These observations are consistent with a model in which exogenous ammonia can access the intramolecular tunnel in AS-B during glutamine-dependent asparagine synthesis, in contrast to expectations based on studies of class I amidotransferases.

EPR and Infrared Spectroscopic Evidence That a Kinetically Competent Paramagnetic Intermediate is Formed When Acetyl-Coenzyme A Synthase Reacts with CO

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme which enables archaea and bacteria to grow autotrophically on CO and hydrogen/carbon dioxide using the Wood-Ljundahl pathway. CO produced from reduction of carbon dioxide by CODH is transferred to the active site of ACS through an intramolecular tunnel, where it combines with Coenzyme A and a methyl cation to produce acetyl-CoA. The active site of ACS contains a single [4Fe-4S] cluster bridged by a cysteine sulfur atom to a binuclear center. The binuclear center is composed of two Ni atoms bridged by two separate cysteine sulfurs. The Ni site attached to the [4Fe-4S] is referred to as proximal Ni, while the other Ni atom, which assumes a square-planar geometry, is referred to as the distal site. We report the characterization of the carbonylated form of highly active (0.67 spins/mol) heterologously expressed monomeric ACS from C. hydrogenoformans in E. coli by rapid-freeze quench EPR (RFQ-EPR) and stopped-flow infrared (SF-IR) spectroscopies. The reaction of ACS with CO produces a single metal-carbonyl species whose formation rate, measured by SF-IR, correlates with the rate of formation, measured by RFQ-EPR, of the paramagnetic state of the enzyme (NiFeC species). These results indicate that the NiFeC species is the predominant form observed in solution when ACS reacts with CO. The NiFeC species contains the proximal Ni in the +1 redox state and the [4Fe-4S] cluster in the 2+ state, thus there is no evidence for either a Ni(0) or a Ni(II) state in the active carbonylated form of the enzyme.

The unexpected structural role of glutamate synthase [4Fe–4S] +1, +2 clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme β subunit

Azospirillum brasilense glutamate synthase (GltS) is a complex iron–sulfur flavoprotein whose catalytically active αβ protomer (α subunit, 162 kDa; β subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe–4S] 0, +1 , and two [4Fe–4S] +1, +2 clusters. The structure of the α subunit has been determined providing information on the mechanism of ammonia transfer from l-glutamine to 2-oxoglutarate through a 30 Å-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe–S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe–4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS β subunit with alanyl residues. The engineered genes encoding the β subunit variants (and derivatives carrying C-terminal His 6-tags) were co-expressed with the wild-type α subunit gene. In all cases the C/A substitutions prevented α and β subunits association to yield the GltS αβ protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe–4S] +1, +2 clusters within the N-terminal region of the β subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in αβ heterodimer formation by structuring an N-terminal [Fe–S] β subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal β subunit-like domain.

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 α subunit (∼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 (β/α) 8 barrel synthase domain connected by (iii) a ∼30 Å-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 (β subunit, ∼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.

Long-range allosteric transitions in carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of carbamoyl phosphate synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at approximately 100 A from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to carbamoyl phosphate synthetase is described.

Glutamate synthase: a fascinating pathway from L-glutamine to L-glutamate.

Glutamate synthase is a multicomponent iron-sulfur flavoprotein belonging to the class of N-terminal nucleophile amidotransferases. It catalyzes the conversion of L-glutamine and 2-oxoglutarate into two molecules of L-glutamate. In recent years the X-ray structures of the ferredoxin-dependent glutamate synthase and of the a subunit of the NADPH-dependent glutamate synthase have become available. Thanks to X-ray crystallography, it is now known that the ammonia reaction intermediate is transferred via an intramolecular tunnel from the amidotransferase domain to the synthase domain over a distance of about 32A. Although ammonia channeling is a recurrent theme for N-terminal nucleophile and triad-type amidotransferases, the molecular mechanisms of ammonia transfer and its control are different for each known amidotransferase. This review focuses on the intriguing mechanism of action and self-regulation of glutamate synthase with a special focus on the structural data.