Glutamate synthase: a fascinating pathway from L-glutamineto L-glutamateR. H. H. van den Heuvela, b, B. Curti c, M. A. Vanonic,* and A. Mattevia,*
a Department of Genetics and Microbiology, University of Pavia, via Abbiategrasso 207, 27100 Pavia (Italy),e-mail: [email protected] Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institutefor Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht (The Netherlands)c Department of Biomolecular Sciences and Biotechnology, University of Milan, via Celoria 26, 20133 Milan (Italy),e-mail: [email protected]
Received 8 August 2003; received after revision 15 September 2003; accepted 17 September 2003
Abstract. Glutamate synthase is a multicomponent iron-sulfur flavoprotein belonging to the class of N-terminalnucleophile amidotransferases. It catalyzes the conversionof L-glutamine and 2-oxoglutarate into two molecules ofL-glutamate. In recent years the X-ray structures of theferredoxin-dependent glutamate synthase and of the asubunit of the NADPH-dependent glutamate synthasehave become available. Thanks to X-ray crystallography,it is now known that the ammonia reaction intermediate is
transferred via an intramolecular tunnel from the amido-transferase domain to the synthase domain over a distanceof about 32 Å. Although ammonia channeling is a recur-rent theme for N-terminal nucleophile and triad-type ami-dotransferases, the molecular mechanisms of ammoniatransfer and its control are different for each known ami-dotransferase. This review focuses on the intriguing mech-anism of action and self-regulation of glutamate synthasewith a special focus on the structural data.
Introduction
In this review we address the fascinating mechanism ofaction of the multicomponent glutamine-dependent ami-dotransferase glutamate synthase, hereafter referred to asGltS, with a special focus on recent structural data. The re-view on GltS of Vanoni and Curti in 1999 [1] in this jour-nal provides the necessary background information on theenzyme and is an excellent introduction for this review.Recent X-ray crystallographic data have revealed that theiron-sulfur and flavin-dependent GltS functions throughthe intramolecular transfer of ammonia between func-tionally distinct domains (namely amidotransferase and
flavin mononucleotide (FMN) binding domains) over adistance of about 32 Å [2–4]. Moreover, GltS is only ac-tive when both L-glutamine and 2-oxoglutarate substratesare bound, the enzyme is in the reduced state and, in thecase of ferredoxin-dependent GltS, in complex with re-duced ferredoxin, hereafter referred to as Fd [5, 6]. Thus,GltS has evolved a sophisticated mechanism to controland coordinate catalytic activities at sites physically dis-tinct from each other, and to channel the reaction inter-mediate between catalytic sites.
Biochemistry of glutamate synthase
Prior to 1970 it was generally assumed that ammonia wasassimilated by direct assimilation of 2-oxoglutarate to* Corresponding authors.
produce L-glutamate in a single NAD(P)H-linked reactioncatalyzed by the enzyme glutamate dehydrogenase. Thepioneering work of Tempest and co-workers [7] demon-strated the existence of a glutamine synthetase/glutamatesynthase pathway, which is operative in bacteria growingon media with a low ammonia content, or in bacteria de-void of glutamate dehydrogenases or equivalent aminoacid dehydrogenases. Further studies revealed that theconcerted action of glutamine synthetase and GltS is re-sponsible for primary and secondary ammonia assimila-tion also in plants (as reviewed in [8] and [9]). The gluta-mine synthetase (GS)/glutamate synthase pathway, alsoknown as the GS/GOGAT pathway from the now obsoleteacronym for GltS, is of crucial importance in microbialand plant cells, as the products L-glutamine and L-gluta-mate are the nitrogen donors for the biosynthesis of major nitrogen-containing compounds, including, for example, other amino acids, nucleotides, chlorophylls,polyamines and alkaloids [10, 11]. GltS is a ubiquitous en-zyme in nature; it has been detected in prokaryotes, ar-chaea and eukaryotes. However the enzyme has not beenfound in higher eukaryotic systems such as Homo sapiens.The enzyme catalyzes the reductant-dependent conversionof 2-oxoglutarate into L-glutamate in which L-glutamine
670 R. H. H. van den Heuvel et al. Glutamate synthase
serves as the nitrogen source for the reaction. The overallcatalytic cycle of GltS can be described by the followingequation in which the reducing equivalents originate fromNADH, NADPH or reduced Fd, depending on the type ofGltS:
The catalytic cycle (equation 1) involves distinct catalyticreactions (fig. 1). After enzyme reduction, L-glutamine ishydrolyzed to yield ammonia and L-glutamate via a glu-tamyl thioester-cysteine intermediate. This reaction is fol-lowed by the addition of ammonia onto 2-oxoglutarate,forming 2-iminoglutarate. 2-Iminoglutarate is then subjectto reduction by a reduced flavin cofactor, forming the sec-ond molecule of L-glutamate.On the basis of the primary structures of GltSs from dif-ferent sources and the known biochemical properties,three distinct classes of GltS can be distinguished [1]:1) Ferredoxin-dependent GltS (Fd-GltS) is an iron-sulfur
and FMN-containing enzyme that has been detected inchloroplasts of higher plants, cyanobacteria and algae.This enzyme is composed of a single polypeptide chainwith a molecular mass of about 165 kDa. The enzymefunctions through the noncovalent binding of a reduced
Figure 1. Schematic representation of the partial reactions catalyzed by GltS leading to glutamine-dependent glutamate synthesis. The am-monia produced in the amidotransferase domain is added onto 2-oxoglutarate in the FMN binding domain. The reducing equivalents orig-inate from reduced ferredoxin (Fd-GltS) or NADPH (through the NADPH b-GltS subunit and the corresponding part of NADH-GltS). Notethat the precise protonation states of intermediates and active site residues are unknown.
Fd molecule, which delivers the reducing equivalentsvia the [3Fe-4S]0,+1 cluster to the FMN prostheticgroup. Biochemical data are available from spinach andSynechocystis sp. Fd-GltS [5, 12, 13], whereas the X-ray structure is known for the Synechocystis enzyme[3, 4].
2) The NADPH-dependent GltS (NADPH-GltS) is mostlyfound in bacteria. The enzyme is composed of twotightly bound dissimilar subunits, which form the abholoenzyme containing one flavin adenine dinu-cleotide (FAD) and one FMN cofactor, and three dis-tinct iron-sulfur clusters: one [3Fe-4S]0,+1 center andtwo low potential [4Fe-4S]+1,+2 clusters. The larger (a)subunit (160 kDa) catalyzes the reductive synthesis ofL-glutamate from L-glutamine and 2-oxoglutarate[14]. The smaller (b) subunit (52 kDa) is a FAD-dependent NADPH oxidoreductase [15]. It serves totransfer reducing equivalents from NADPH to theFMN cofactor (on the a subunit) through the FAD cofactor (on the b subunit) and at least two of theNADPH-GltS three iron-sulfur clusters, namely: the[3Fe-4S]0,+1 and one of the two low potential [4Fe-4S]+1,+2 centers of the enzyme. Most of the infor-mation on this class of enzymes derives from workdone in our laboratories on the Azospirillum brasilenseenzyme. The three-dimensional structure of the a sub-unit of this enzyme has been solved [2].
3) NADH-dependent GltS is poorly characterized and ismainly found in fungi, lower animals and nongreen tis-sues like seeds and roots of plants. The enzyme is com-posed of a single polypeptide chain of about 200 kDa.Sequence analyses suggest that the enzyme is derivedfrom a fusion of the genes coding for the a and b sub-units of the NADPH-dependent enzyme. UnlikeNADPH-GltS, the eukaryotic pyridine nucleotide-de-pendent form of GltS is highly specific for NADH asthe electron donor.
GltS is a member of the glutamine-dependent amido-transferase family of enzymes. These enzymes catalyzethe transfer of the amide group of L-glutamine, the sourceof nitrogen for most biosynthetic pathways, to an accep-tor substrate, which is different for each glutamine-dependent amidotransferase, to produce one molecule ofL-glutamate and one molecule of aminated (or amidated)product. To date 13 different glutamine-dependent ami-dotransferases have been identified [16–19]. The enzymesare involved in the biosynthesis of nucleotides, aminoacids, aminated sugars and antibiotics. All amidotrans-ferases exhibit a modular organization with physically dis-tinct catalytic centers for L-glutamine hydrolysis and theaddition of ammonia to the acceptor substrate, respec-tively, which are connected by an intramolecular ammoniatunnel. The amidotransferase family can be divided in fourdifferent classes: the N-terminal nucleophile (Ntn), thetriad, the amidase and the nitrilase class [17, 20, 21]. The
amidase and nitrilase family are only poorly characterized.Functionally, the main differences between the Ntn andtriad type are the location of the catalytic cysteine residueand the mechanisms of activation of the cysteinyl thiol. Inthe triad amidotransferases the catalytic cysteine is foundto be part of the catalytic triad Cys-His-Glu in which theHis and Glu residues are postulated to activate the cysteinethiol group. In the Ntn-type amidotransferases the cat-alytic cysteine is located at the N-terminus of the matureprotein, and activation of the cysteinyl thiol is assisted bythe free amino-terminal group [19]. Furthermore, no sim-ilarity between amidotransferases of the different classesis found at the primary and tertiary structure level. Finally,as we shall discuss later, both the intramolecular tunnel forammonia transfer and the synthase function are unrelatedto each other in different amidotransferases even withineach one of the four known classes of enzymes. GltSs areunique among Ntn-type amidotransferases in that theycatalyze an oxidoreduction reaction, which appears tocontribute a further layer of both complexity and controlover the catalytic activity of the enzymes.
The glutamate synthase structure
In this review we focus on Fd-GltS from Synechocystis sp.and NADPH-GltS from A. brasilense as the two best-stud-ied GltSs. Fd-GltS and the a subunit of NADPH-GltS arerather similar enzymes of about 160–165 kDa as also in-dicated by the high amino acid sequence identity (45%).Crystal structures of A. brasilense GltS a subunit and ofseveral forms of Fd-GltS from Synechocystis sp. are avail-able (table 1). Here, we shall mainly focus on the Fd-GltSstructures since they have been solved at higher resolu-tion.The tertiary structures of the a subunit of NADPH-GltSand Fd-GltS, as determined by X-ray crystallography, aresimilar as also indicated by the root mean square deviationof 1.7 Å for all topological equivalent Ca atoms. EachGltS monomer can be described in terms of four distinctdomains with different function and topology (fig. 2).Doolittle and co-workers screened 16 strains from Ther-motoga and other related Thermotogales for the occur-rence of the archaeal a subunit of GltS. The results clearlyshowed that the different domains in GltS are indeed re-cruited from preexisting domains [22]. The N-terminalamidotransferase domain (residues 1–422 according toFd-GltS numbering, which will be used throughout unlessotherwise stated) is characterized by a classical four-layera/b/b/a topology similar to that of the three other char-acterized Ntn-type amidotransferases. Indeed, super-positions of Fd-GltS onto glutamine phosphoribosylpy-rophosphate amidotransferase (root mean squaredeviation 2.5 Å for 217 Ca atoms) [23], glucosamine 6-phosphate synthase (root mean square deviation 2.9 Å for
217 Ca atoms) [24] and asparagine synthetase B (rootmean square deviation 5.2 Å for 185 Ca atoms) [25] re-veal close structural similarity.The FMN binding domain consists of residues787–1223 and forms, next to the amidotransferase do-main, the second catalytic domain in GltS. This domainhas a typical (b/a)8 barrel topology and contains the [3Fe-4S]0,+1 cluster and the FMN prosthetic group of theenzyme. The (b/a)8 barrel of the GltS FMN binding domain resembles that of other FMN binding enzymes.The most similar structures as found by DALI [26] are theFMN binding domains of flavocytochrome b2 (root meansquare deviation 3.1 Å for 254 Ca atoms) [27], glycolate
oxidase (root mean square deviation 3.2 Å for 278 Caatoms) [28] and dihydroorotate dehydrogenase (root meansquare deviation 3.2 Å for 221 Ca atoms) [29]. The high-est level of structural similarity is found in the core of thisdomain, which is the FMN binding region. A BLAST homology search revealed a unique peptide stretch in Fd-GltS, absent in NADPH-GltS a subunit and the cor-responding part of NADH-GltS. These 26 residues(residues 907–933), located on the surface of the proteinin the vicinity of the [3Fe-4S] cluster and the FMN co-factor, are highly conserved in all Fd-GltSs. We have pro-posed that reduced Fd can bind near this peptide stretch(hereafter referred to as the Fd-binding loop) to transfer anelectron via the [3Fe-4S] cluster to the FMN for 2-iminog-lutarate reduction [3]. To our best knowledge, Fd-GltS isthe only Fd-dependent enzyme in which such a potentialFd-binding loop is recognized.Like the FMN binding domain, the central domain(residues 423–786) is characterized by an b/a topology.Binda and co-workers [2] have demonstrated that this domain is an extended half (b/a)8 barrel as its core consists of five consecutive b/a units that generate a par-allel b sheet flanked on one side by four a helices and onthe other side by a single a helix running perpendicular to the sheet axis. Remarkably, DALI [26] revealed highstructural similarity with the FMN-binding enzymes glycolate oxidase and dihydroorotate dehydrogenase (root mean square deviations 2.6 Å for 189 Ca atoms and3.7 Å for 179 Ca atoms, respectively). In addition, in spite of the low sequence identity with the GltS FMNbinding domain, the central and FMN binding domainsare structurally similar [2]. Structural similarity was alsoidentified with the HisF protein fragment of imidazoleglycerol phosphate synthase (root mean square devia-tion 3.2 Å for 123 Ca atoms) [30]. HisF consists of a(b/a)8 barrel with a twofold repeat pattern. It is postulatedand verified by protein engineering that HisF has evolvedby a twofold gene duplication and gene fusion from acommon half-barrel ancestor [30, 31]. A fascinating ques-tion is whether also the extended half (b/a)8 barrel of theGltS central domain derives from an ancient half (b/a)8
barrel.
672 R. H. H. van den Heuvel et al. Glutamate synthase
Table 1. Known X-ray structures of glutamate synthase.
a GltS from A. brasilense.b GltS from Synechocystis sp.c The FMN cofactor in the GltS crystallographic model is in the reduced state.
Figure 2. Tertiary structures of the Fd-GltS and Fd monomers. TheN-terminal amidotransferase domain is depicted in cornflower blue,the FMN-binding domain in yellow, the central domain in magentaand the C-terminal domain in green. The Fd molecule is depicted inred. The FMN cofactor and the iron-sulfur clusters are shown asball-and-stick, the Fd-binding loop in red and the cavities in grey.The position of Fd relative to Fd-GltS is not based on experimentalevidence. The surface of active site cavities and the internal ammo-nia tunnel are shown. This drawing was prepared with CONSCRIPT[47] and Raster3D [48].
The C-terminal GltS residues 1224–1523 form a right-handed b helix that comprises seven helical turns. Thecross-section of the b helix has an ellipsoidal shape, andthe helix axis extends for 43 Å. A DALI [26] search re-vealed that the structural similarity of this domain withother b helices is only remote. The highest structural sim-ilarity was found with polygalacturonase from Erwiniacarotovora [32] and pectate lyase from Bacillus sp. [33](root mean square deviation of 4.1 Å for 150 residues and3.2 Å for 126 residues, respectively). In contrast to the C-terminal domain in GltS, these b helices in polygalactur-onases perform a catalytic reaction, namely the hydroly-sis of the a-1,4-glycosidic bond in polygalacturonic acids.The role of the C-terminal b helix in GltS is unknown.However, recent modeling studies, based on synchrotronradiation X-ray scattering measurements on solutions ofNADPH-GltS a subunit, indicate that in this enzyme theC-terminal b helix may contribute to tetramerization of asubunits by interacting with the glutamine amidotrans-ferase domain of the adjacent one [34].
Quaternary structure of glutamate synthase
Small-angle X-ray scattering data have provided evidencethat Fd-GltS is a monomeric protein in solution [4]. Incontrast, nanoflow electrospray ionization mass spec-trometry experiments of Fd-GltS revealed that the proteinis in equilibrium between the monomeric and dimericform with m/z values around 7000 and 9000, respectively(fig. 3) [A. J. R. Heck and R. H. H. van den Heuvel, un-published]. The molecular mass of Fd-GltS monomer de-termined by mass spectrometry is 166,222 Da, which is inclose agreement with the mass calculated on the basis ofthe amino acid sequence of the mature holoprotein(166,230 Da) considering the noncovalently bound FMNcofactor (457 Da) and the [3Fe-4S] cluster (296 Da). TheX-ray data of Fd-GltS have shown that the enzyme isdimeric in the crystalline state [3, 4]. We have to take intoaccount, however, that the results obtained by any of thethree techniques cannot be directly related to each other,although several reports have revealed that no major dif-ferences are to be expected at least when working at thesame protein concentration, pH and ionic strength. Dis-crepancies in the oligomerization state were also found forthe a subunit of NADPH-GltS. Small-angle X-ray scat-tering experiments indicated tetrameric assemblies of thea subunit of NADPH-GltS [34], whereas X-ray analysisof the crystalline protein clearly reveals dimers [2]. On thecontrary, a good match between the oligomerization stateof NADPH-GltS ab holoenzyme determined by small-an-gle X-ray scattering [34] and analytical gel filtration [36]has been observed. Indeed, both techniques indicate that the enzyme forms tetramers at concentrations above 5 mM.
For Fd-GltS to produce L-glutamate from L-glut-amine and 2-oxoglutarate, reducing equivalents originat-ing from reduced Fd are necessary. Synchrotron radiationX-ray scattering data have shown that Fd-GltS forms anequimolar complex with Fd [4]. The Fd:Fd-GltS complexwas also studied by electrospray ionization mass spec-trometry, confirming that the two proteins form anequimolar complex (fig. 3). Even when we added a three-fold molar excess of Fd to Fd-GltS, we only observed a1:1 complex with a molecular mass of 176,698 Da,which is in close agreement with the expected mass of a1:1 complex (176,735 Da including the FMN, [3Fe-4S]and [2Fe-2S] noncovalently bound cofactors). The Fd:Fd-GltS assembly appeared to interact very tightly as themass spectrometric data of a 1 mM equimolar mixture of
Figure 3. Nanoflow electrospray ionization mass spectra in positiveion mode of native folded Fd-GltS and the complex between Fd-GltS and Fd sprayed from 50 mM ammonium acetate, pH 6.8. Top: The mass spectrum of 1 mM Fd-GltS reveals the monomericprotein at m/z around 7000 (mean charge state +24) and dimeric pro-tein at m/z around 9000 (mean charge state +36). Bottom: The massspectrum of 1 mM Fd-GltS mixed with 1 mM Fd reveals the equimo-lar complex between the proteins in both their monomeric form atm/z around 7000 (mean charge state +25) and dimeric form at m/zaround 9500 (mean charge state +37). The insets show the zoomed-in part of m/z 6800–7200 and display the relative ion abundance ofGltS [M+24H]+ and Fd:Fd-GltS [M+25H]+. The most abundant ionpeak of the heterodimeric complex between Fd and Fd-GltS has onecharge more than the most abundant peak of Fd-GltS.
m/z
6800 7000 7200
6800 7000 7200R
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ive
abun
danc
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abun
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the two proteins revealed nearly complete complex for-mation (> 90%).
Glutaminase reaction of glutamate synthase
The glutaminase site in the amidotransferase domain cat-alyzes the hydrolysis of L-glutamine into the first mole-
cule of L-glutamate and ammonia. Ammonia is then trans-ferred to the second catalytic center, where it is added onto2-oxoglutarate (fig. 1). The fold of the Ntn-type amido-transferases is the signature of the structural superfamilyof Ntn hydrolases that appear to be evolutionary related,but have diverged beyond any recognizable primary struc-ture similarity [37]. The substrate binding pocket, theoxyanion hole, the proton donor and the catalytic nucle-
674 R. H. H. van den Heuvel et al. Glutamate synthase
Figure 4. Comparison of the glutaminase site in Ntn-typeamidotransferases. (A) Superposition of the substrate bindingpocket of 5-oxo-L-norleucine-Cys1 complexed Fd-GltS (darkorange), 5-oxo-L-norleucine-Cys1 complexed glutaminephosphoribosylpyrophosphate amidotransferase (yellow), un-liganded glucosamine-6-phosphate synthase (light grey) andL-glutamine liganded aspargine synthetase B (dark grey). Theseven amino acid residues depicted are conserved within Ntn-type amidotransferases. (B) Superposition of the glutaminasesite of the 5-oxo-L-norleucine-Cys1 complexed GltS (dark or-ange) and the L-methionine sulphone liganded aGlts (blue).This stereo view drawing was prepared with MOLSCRIPT[49].
A
B
ophile occupy topological equivalent sites in all membersof this superfamily. The mechanism of action of L-gluta-mine hydrolysis in Ntn-type amidotransferases, as dis-cussed below, appears to be similar to that of other Ntn hy-drolases even though the nucleophile varies: cysteine inamidotransferases, serine in penicillin acylase and threo-nine in 20S proteasome and aspartylglucosaminidase. Thefree N-terminus is thought to be essential for activation ofthe active site nucleophile (Cys in amidotransferases) andprotonation of the leaving group (ammonia in amido-transferases) in the hydrolytic reaction. Moreover, the freeN-terminal amino group should participate in deprotona-tion of the water molecule required to hydrolyze the glutamyl-thioester intermediate [19].The four members of the Ntn amidotransferase family,GltS [2–4], asparagine synthetase B [25], glutamine phos-phoribosylpyrophosphate amidotransferase [23, 38–41]and glucosamine 6-phosphate synthase [24, 42] have beenthoroughly characterized, and for all enzymes one or moreX-ray structures are known in the free state and/or in com-plex with substrate (analogues), product or inhibitors. Thesubstrate binding pockets of the four Ntn amidotrans-ferases are remarkably well conserved (fig. 4A). 5-Oxo-L-norleucine in GltS and glutamine phosphoribosylpy-rophosphate amidotransferase [39] and L-glutamine in theCys1Ala mutant of asparagine synthetase B [25] bind inequivalent positions. L-glutamine is specifically recog-nized by the glutaminase binding pocket forming an ex-tensive hydrogen bond network with the invariant residueslining the catalytic centre: Cys1, Arg31, Arg206, Thr209,Asn227, Gly228 and Asp270 (numbering according to Fd-GltS) (fig. 4A). The substrate is oriented such that theamide group points with its carbonyl oxygen towards theoxyanion hole (Asn227 and Gly228), its carbon atom is incorrect juxtaposition to the nucleophilic sulfur atom ofCys1 and the amide leaving group is within reach of theamino terminus, which acts as the proton donor. The ini-tial binding of the L-glutamine substrate is thought to befollowed by closure of the glutamine lid loop (residues206–214, also known as Q-loop), which protects L-glut-amine from bulk solvent. The substrate lid loop in GltSappears to be in the open conformation [2, 3]. Within thisloop, only the position of Thr209 is not conserved in GltSwith respect to its three homologues in which the gluta-mine lid loop is (partially) closed (fig. 4A). GltS has beencrystallized in both the catalytically active (all structuresof Fd-Glts; table 1) and inactive conformations (structureof a-GltS in complex with L-methionine sulphone;table 1) (fig. 4B). By comparing these structures, it ap-pears that the binding of the substrate analogue L-me-thionine sulphone shifts the position of Cys1 and loop29–34 out of the substrate-binding pocket so that the en-zyme becomes catalytically incompetent [4].The reaction mechanism by which L-glutamine is hy-drolyzed to glutamate and ammonia is thought to occur
via nucleophilic attack on the substrate carbonyl atom bythe Cys1 thiolate anion (fig. 4A). This initial nucleophilicattack leads to the formation of a covalently bound glu-tamyl thioester intermediate, which is mimicked by the 5-oxo-L-norleucine-Cys1 adduct in some of the GltS andglutamine phosphoribosylpyrophosphate amidotrans-ferase structures. Indeed, the position of 5-oxo-L-nor-leucine in the substrate binding pocket is consistent withthe proposed mechanism of glutamine hydrolysis [4, 23].The final product L-glutamate has been cocrystallizedwith the isolated amidotransferase domain of glucosamine6-phosphate synthase [42], which is, however, not cat-alytically competent. However, the position and hydrogenbond interactions of L-glutamate appear to be conservedwith respect to the position and hydrogen bond pattern ofL-glutamine. The intermediate ammonia product must bekept within the enzyme and gated through an intramolec-ular tunnel to the synthase site where it is added to an ac-ceptor substrate which differs for each of the amidotrans-ferases: 2-oxoglutarate for GltS (fig. 1), fructose 6-phosphate for glucosamine 6-phosphate synthase, aspar-tate for asparagine synthetase B and 5-phosphoribosyl-(a)1-pyrophosphate for glutamine phosphoribosylpy-rophosphate amidotransferase.
Synthase reaction of glutamate synthase
In the FMN binding domain of GltS, ammonia, originat-ing from the glutaminase site, is added onto 2-oxoglu-tarate to form the 2-iminoglutarate reaction intermediate.This intermediate product is then reduced by the FMN co-factor to yield the second molecule of L-glutamate(fig. 1). The X-ray models of the FMN binding domainsof NADPH-GltS and Fd-GltS are highly similar [2, 3].Catalysis in the synthase site is formally initiated by thebinding of 2-oxoglutarate. The 2.0-Å-resolution X-raystructure of GltS in complex with 2-oxoglutarate hasshown unambiguously the positions of all the substrateatoms (fig. 5A). We have demonstrated before that thecarbonyl oxygen of 2-oxoxglutarate is stabilized byLys972 and that the substrate is ideally positioned to re-ceive the ammonia molecule from the intramolecular tun-nel [4]. Furthermore, the observed position of 2-oxoglu-tarate does not allow its reduction by the reduced FMNcofactor, since the distance between C2 of 2-oxoglutarateand N5 of FMN is longer than 4.0 Å and the 2-oxoglu-tarate C1-C2(O)-C3 plane is not parallel with the FMNisoalloxazine ring. Insights in catalysis of reductive synthesis of L-glutamateat the FMN binding domain of GltS could be obtained incomparison with the well-characterized flavocytochromeb2. Flavocytochrome b2 catalyzes the oxidation of L-lac-tate to pyruvate with the subsequent transfer of electronsto cytochrome c. Thus, the stereochemistry of the GltS-
catalyzed reaction is the same as that of flavocytochromeb2. The enzyme is a homooctamer with a subunit molec-ular mass of 57.5 kDa containing two noncovalentlybound cofactors, FMN and heme, per subunit [43]. Itsthree-dimensional structure in complex with the reactionproduct pyruvate has been determined [27]. Pyruvate isbound parallel to the FMN isoalloxazine ring, and theshortest distance between cofactor and product (3.5 Å) in-volves the flavin N5 atom and the pyruvate carbonylC2 atom. This position of pyruvate fulfills all require-ments for enzyme catalysis. Superposition of the FMNbinding domains of GltS and flavocytochrome b2 clearlyreveals the differences between binding of their respectiveligands. We have argued that the addition of ammonia to2-oxoglutarate initiates a rotation of the 2-iminoglutarateproduct such that this intermediate is properly positionedfor reduction by reduced FMN. Thus, the C1-C2(N)-C3 plane is likely to be planar with the FMN isoalloxazinering, and the distance between C2 2-iminoglutarate andN5 FMN should be about 3.5 Å [4], a conformation sim-ilar to that of pyruvate in flavocytochrome b2. Superposi-tion of a modeled 2-iminoglutarate in the active site ofGltS and the X-ray model of pyruvate-liganded flavocy-tochrome b2 indicates the homology between the two en-zyme active sites (fig. 5B). Mathews and co-workers havepostulated that the hydroxyl moiety of L-lactate is hydro-gen bonded to His373 and Tyr254, as is the carbonyl oxy-
gen in the pyruvate product [27]. The residues equivalentto Tyr254 and His373 of flavocytochrome b2 areGlu903 and Gln969 in GltS, respectively, with their sidechains pointing in the active site cavity. Indeed, the iminenitrogen of the modeled 2-iminoglutarate may form a hydrogen bond interaction with the side chains ofGlu903 and Gln969.Thus, GltS catalyzes ammonia addition to 2-oxoglutarateand reduction of 2-iminoglutarate in one active site. Thecharged residues Lys972 and Glu903 appear to be crucialfor this dual functionality as Lys972 anchors the carbonyloxygen of 2-oxoglutarate, fixing the substrate in an ori-entation that is suited for ammonia addition, but not for re-duction by FMN. Lys972 is likely to play a prominent rolein catalysis through polarization of the C2 atom, makingthis atom more prone to nucleophilic attack by ammonia.After 2-iminoglutarate formation, the negative charge ofthe side chain of Glu903 fixes the intermediate in theproper orientation for reduction by FMN, allowing for-mation of the second molecule of L-glutamate.
Ammonia tunnel
The X-ray structures of GltS reveal that the glutaminasesite and the synthase site are separated by more than 30 Ådistance. An internal ammonia tunnel connects the two ac-
676 R. H. H. van den Heuvel et al. Glutamate synthase
Figure 5. Synthase active site in the FMN binding domain of GltS. (A) Superposition of 2-oxoglutarate-liganded Fd-GltS and pyruvate-liganded flavocytochrome b2. The carbonyl oxygen of 2-oxoglutarate is hydrogen bonded to Lys972 and Gln978, whereas the carbonyl oxy-gen of pyruvate is hydrogen bonded to Tyr254 and His373. (B) Superposition of the modeled 2-iminoglutarate-liganded Fd-GltS and of pyru-vate-liganded flavocytochrome b2. The imino group of 2-iminoglutarate now potentially interacts with Glu903 and Gln969. The latter tworesidues are spatially equivalent to Tyr254 and His373 in flavocytochrome b2. This drawing was prepared with MOLSCRIPT [49] andRaster3D [48].
A B
tive sites (fig. 2). The tunnel entrance is located close tothe amidotransferase active site, at the interface amongthree domains, namely the amidotransferase domain, thecentral domain and the C-terminal domain. In differentGltS structures, there are no detectable differences in thegeometry of the tunnel itself, nor of residues restricting itat either glutaminase or synthase sites. The tunnel pointsinto the FMN binding domain and mainly consists ofresidues from the FMN binding domain. A major part ofone wall of the tunnel is formed by residues 1003–1013,
which belong to loop 4 (residues 968–1013) of the FMN-binding (b/a)8 barrel [2, 3].Comparison of the GltS ammonia tunnel with the tunnelsin glucosamine 6-phosphate synthase, asparagine syn-thetase B and glutamine phosphoribosylpyrophosphateamidotransferase reveals marked differences among thefour enzymes as also recently pointed out by Raushel andco-workers [44]. The 32-Å-long GltS tunnel consistsmainly of backbone atoms of hydrophilic residues, but italso involves the side chains of Glu903, Ser947 and
Figure 6. Cartoon of hydrogen bond interactions between ordered water molecules within the GltS ammonia tunnel and the residues lin-ing the tunnel. The 2-oxoglutarate substrate is depicted as ball-and-stick and the water molecules as spheres.
Lys966 [2, 3]. We found several ordered water moleculesin the tunnel interacting with residues forming the tunnelvia an extensive hydrogen bond network (fig. 6). The wa-ter molecules completely fill up the tunnel space, and itseems likely that their positions change upon ammoniatransfer during catalysis.The 20-Å-long ammonia tunnel of glutamine phosphori-bosylpyrophosphate amidotransferase is lined with non-polar residues and consists of a string of small cavities be-tween the active sites, suggesting that conformationalbreathing of the tunnel is necessary for ammonia chan-neling. By solving the active and inactive enzyme X-raystructure, Smith and co-workers have demonstrated thatthe tunnel is formed only transiently during catalysis[23, 40]. When the enzyme is crystallized in the presenceof 6-diazo-5-oxo-L-norleucine and a carbocyclic analogueof phosphoribosylpyrophosphate, creating a state whichmimics the enzyme just after ammonia release and withsubstrate bound in the synthase site, the C-terminal helixbecomes extensively kinked and the flexible loop becomesordered forming a molecular tunnel [23]. Like glutaminephosphoribosylpyrophosphate amidotransferase, the am-monia tunnel of glucosamine 6-phosphate synthase israther hydrophobic and has a length of about 20 Å [24]. Inthe reported X-ray structure of the holoenzyme the tunnelis accessible for bulk solvent; however, it is speculated thatthe glutamine lid loop, which covers the active site whenproduct L-glutamate and substrate analog L-glutamate-g-hydroxamate are bound to the isolated amidotransferasedomain [42], closes and isolates the tunnel from solventupon L-glutamine substrate binding. The length of themolecular tunnel in asparagine synthetase B is also about20 Å and is formed primarily by backbone atoms and hy-drophobic or nonpolar residues. However, the side chainof a glutamate residue is identified at the end of the tun-nel near the AMP moiety [25]. The X-ray structure of thisenzyme in the presence of L-glutamine and adenosylmonophosphate revealed, as in GltS, several ordered wa-ter molecules within the ammonia tunnel.The observed differences in the molecular structures ofthe tunnels are not surprising as most of the tunnel isformed in all cases by the synthase domains of the en-zymes, which are not related to each other. In GltS, thesynthase domain is the FMN binding domain with an(a/b)8 topology. The isomerase domain of glucosamine 6-phosphate synthase has a canonical nucleotide-bindingfold. The C-terminal domain of asparagine synthetase B ischaracterized by a five-stranded parallel b sheet flankedon either side by a helices. Finally, the phosphoribosyltransferase domain of glutamine phosphoribosylpy-rophosphate amidotransferase consists of a parallel bsheet enclosed by a helices. On the other hand, even insynthase domains with equivalent topologies, ammoniatransfer can occur via different molecular mechanisms.For instance, imidazole glycerol phosphate synthase is a
triad-type amidotransferase which catalyzes the formationof the imidazole ring in histidine biosynthesis. As in GltS,the synthase domain, harbored in the HisF subunit, ex-hibits a classical (b/a)8 domain topology. However, atvariance with GltS, the active site is on the barrel side farfrom the amidotransferase site, so that the tunnel of imi-dazole glycerol phosphate synthase runs across the barrelinterior [45, 46]. This differs significantly from the tunnelin GltS that runs under loop 4 extending from the C-ter-minal part of the barrel, where the synthase active site islocated.
Enzyme self-regulation
GltS functions through hydrolysis of L-glutamine in theglutaminase site into L-glutamate and ammonia. Ammo-nia is then transferred through the intramolecular tunnel tothe synthase site where it is added to 2-oxoglutarate. Theresulting 2-iminoglutarate is reduced by the reduced FMNcofactor, producing the second molecule of L-glutamate(fig. 1). The FMN cofactor in Fd-GltS receives its elec-trons, via the [3Fe-4S] cluster, from two molecules of re-duced Fd. How is the glutaminase activity in Fd-GltS reg-ulated such that L-glutamine is only hydrolyzed when theacceptor substrate 2-oxoglutarate and the reduced electrondonating protein Fd are present?It was proposed earlier that GltS regulates its activity byusing a unique mechanism of self-regulation in whichloop 4 (residues 968–1013) of the FMN (b/a)8 barrelplays an important role [2, 4]. The N-terminal region ofloop 4 is in the vicinity of the loop carrying the [3Fe-4S]cluster and of the Fd-binding loop (residues 907–933).Loop 4 is also involved in binding of 2-oxoglutaratethrough residues Arg992, Lys972 and Gln969, whereas atthe C-terminus loop 4 interacts with the catalytic residueCys1. Figure 7 presents a model of the mechanism of ac-tion of Fd-GltS. It is proposed that upon binding of 2-ox-oglutarate and of reduced Fd in Fd-GltS, loop 4 shiftsfrom an inactive conformation to the active state in whichits C-terminal residue Glu1013 forms a hydrogen bondwith Cys1. This interaction activates the glutaminase sitesuch that L-glutamine can bind in the active site cavity.Upon binding of L-glutamine, loop 206–214 is likely toclose to prevent solvent molecules from entering the sub-strate-binding pocket. Now, L-glutamine is hydrolyzed,forming L-glutamate and ammonia. The closed substratebinding pocket shields ammonia from solvent, and there-fore ammonia may reach the tunnel without being proto-nated. Ammonia is transferred from the glutaminase siteto the synthase site in a diffusion-controlled manner, afterwhich it is added onto 2-oxoglutarate, producing the 2-iminoglutarate intermediate. The latter rotates such that itbecomes properly positioned to be reduced by the reducedFMN prosthetic group (fig. 5B). In the final step 2-
678 R. H. H. van den Heuvel et al. Glutamate synthase
iminoglutarate is reduced by FMN and the second mole-cule of L-glutamate is released. As discussed previously,Met475 may also contribute to the complex regulation ofGltS activity [2]. This residue is in contact with both the[3Fe-4S] cluster and the FMN cofactor, and it may sensethe cofactor’s redox state, transmitting such a signal to theamidotransferase domain and the ammonia tunnel.In conclusion, enzyme self-regulation and ammoniachanneling are common solutions found in all amido-transferases for efficient catalysis without wasteful con-sumption of substrates and intermediates and to transferthe amide nitrogen from L-glutamine to the acceptor sub-strate. However, while amidotransferase domains are con-served and fall in the two well characterized triad and Ntn-type classes of enzymes, both the properties of ammoniatunnels and the mechanisms of control and coordinationof catalysis at the glutaminase and synthase sites differamong the known glutamine-dependent amidotrans-ferases. Furthermore, the acceptor domains are unrelatedto each other. These observations indicate that amido-transferases have evolved independently from each otherthrough recruitment of preexisting protein functions. Inthe case of amidotransferases, it appears to have been aneasier task for the enzymes to recruit and optimize sepa-rately two enzymatic functions, and design a means totransfer the reaction intermediate, than to accommodate
the ancestral separate enzymatic functions in a singlefunctional active site. In light of the properties of ammo-nia, for which, at variance with, for example, acetylgroups in lipoate-dependent enzymes or CO2 in biotin-de-pendent enzymes there is no carrier, intramolecular tun-nels seem to have been the successful common solutionfound for its transfer in all these enzymes.
Acknowledgements. R. H. H. van den H. was supported by a MarieCurie Fellowship (HPMF-CT-2000–00786) from the EuropeanCommunity. This work was supported by grants from the Ministerodella Università e Ricerca Scientifica e Tecnologica (Progetti FIRB,PRIN01 and Legge 449/97). We thank A. Coda and F. J. Florenciofor helpful discussions and support during the glutamate synthaseproject.
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