Structural basis for the catalytic mechanism and�-ketoglutarate cooperativity of glutamate dehydrogenaseReceived for publication, October 5, 2017, and in revised form, March 11, 2018 Published, Papers in Press, March 14, 2018, DOI 10.1074/jbc.RA117.000149

Prem Prakash, Narayan S. Punekar, and Prasenjit Bhaumik1

From the Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai-400076, India

Edited by Ruma Banerjee

Glutamate dehydrogenase (GDH) is a key enzyme connectingcarbon and nitrogen metabolism in all living organisms. Despiteextensive studies on GDHs from both prokaryotic and eukary-otic organisms in the last 40 years, the structural basis of thecatalytic features of this enzyme remains incomplete. This studyreports the structural basis of the GDH catalytic mechanism andallosteric behavior. We determined the first high-resolutioncrystal structures of glutamate dehydrogenase from the fungusAspergillus niger (AnGDH), a unique NADP�-dependent allos-teric enzyme that is forward-inhibited by the formation of mixeddisulfide. We determined the structures of the active enzymein its apo form and in binary/ternary complexes with boundsubstrate (�-ketoglutarate), inhibitor (isophthalate), coenzyme(NADPH), or two reaction intermediates (�-iminoglutarateand 2-amino-2-hydroxyglutarate). The structure of the for-ward-inhibited enzyme (fiAnGDH) was also determined. Thehexameric AnGDH had three open subunits at one side andthree partially closed protomers at the other, a configura-tion not previously reported. The AnGDH hexamers havingsubunits with different conformations indicated that its �-ketoglutarate– dependent homotropic cooperativity followsthe Monod–Wyman–Changeux (MWC) model. Moreover, theposition of the water attached to Asp-154 and Gly-153 definedthe previously unresolved ammonium ion-binding pocket, andthe binding site for the 2�-phosphate group of the coenzyme wasalso better defined by our structural data. Additional structuraland mutagenesis experiments identified the residues essentialfor coenzyme recognition. This study reveals the structuralfeatures responsible for positioning �-ketoglutarate, NADPH,ammonium ion, and the reaction intermediates in the GDHactive site.

Enzymes are important biological macromolecules, and theircatalytic functions govern a number of biological activities in allliving organisms. Visualization of the active site of an enzyme–substrate complex or an enzyme bound to the catalyticallycompetent reaction intermediate provides direct proof of the

reaction mechanism (1). Allosteric regulation of enzymes is oneof the most fundamental processes that control several cellularactivities. Obtaining quantitative molecular description ofenzyme allostery has remained a central focus in biology (2).However, trapping the various structural intermediate states togain detailed understanding about the kinetic properties ofallosteric enzymes has remained very challenging (2, 3). Gluta-mate dehydrogenase (GDH)2 is an oxidoreductase importantfor ammonia metabolism in archebacteria, eubacteria, andeukaryotes (4, 5). We have extensively studied this enzyme todiscern the structural basis of unique properties related to itscatalytic mechanism and allosteric behavior. GDH catalyzes thereversible oxidation of L-glutamate to �-ketoglutarate andserves as a coupler between carbon and nitrogen metabolism.Depending on the coenzyme specificity, GDHs can be classifiedas follows: (a) NADP�-dependent; (b) NAD�-dependent; and(c) NAD�/NADP�-dependent (or dual-specific) (6 –8). Gener-ally, the GDHs involved in ammonium assimilation areNADP�-specific, whereas the NAD�-dependent enzymes areinvolved in glutamate catabolism (9). The mammalian GDHspossessing dual specificities can use either NAD� or NADP�

with comparable efficiency and are allosterically regulated (10,11). NADP� and NAD� are identical except that NADP� hasan extra phosphate group attached to the 2�-hydroxyl of theadenosine. This specific difference is structurally remote to thereactive nicotinamide groups of these two coenzymes. Accord-ingly, the redox potentials of these two coenzymes are almostidentical (8). Nature has developed multiple classes of GDHsthat can efficiently discriminate between these two coenzymes,but how they accomplish this differentiation is unclear.

The NADP�-specific bacterial/fungal GDHs and the dualcoenzyme-specific mammalian GDHs are hexameric (12). TheNAD�– dependent bacterial/fungal enzymes are either homo-hexamers (13) or homotetramers (14, 15). Despite numerousstudies in the last 40 years to identify the intermediates formedduring the reaction catalyzed by GDHs (16 –20), the structuralbasis of the reaction mechanism of this enzyme remains unre-solved. A number of medium/low-resolution crystal structuresof GDHs have been determined as complexes with substrates(�-ketoglutarate or glutamate) and coenzymes (NADP� or

This work was supported by Ramalingaswami Re-entry Fellowship (Depart-ment of Biotechnology, Ministry of Science and Technology, India) and aresearch seed grant from IRCC, IIT Bombay (to P. B.). The authors declarethat they have no conflicts of interest with the contents of this article.

This article contains Figs. 1–10, Table 1, and supporting Refs. 1–3.The atomic coordinates and structure factors (codes 5XVI, 5XVX, 5XVV, 5XWC, and

5XW0) have been deposited in the Protein Data Bank (http://wwpdb.org/).1 To whom correspondence should be addressed. Tel.: 91-22-2576-7748; Fax:

91-22-2572-3480; E-mail: pbhaumik@iitb.ac.in.

2 The abbreviations used are: GDH, glutamate dehydrogenase; fiAnGDH;forward-inhibited A. niger GDH; MWC, Monod–Wyman–Changeux; AKG,�-ketoglutarate; AIG, �-iminoglutarate; AHG, 2-amino-2-hydroxygl-utarate; IPT, isophthalate; PDB, Protein Data Bank; r.m.s.d., root meansquare deviation; KNF, Koshland-Nemethy-Filmer; 2-HED, 2-hydroxy-ethyl disulfide.

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NAD�) (7, 12, 13, 16, 21–23). However, these structures fail toexplain the reaction mechanism and the role of different active-site residues in catalysis, mainly due to their limited resolutionand unavailability of any intermediate-bound, catalyticallycompetent structures of this enzyme. The binding site ofammonia has yet to be identified in the GDH active site. Struc-tural determinants for the recognition of NADP� in NADP�-specific GDHs have remained ambiguous. Significantly, no rep-resentative structure of a fungal enzyme is available so far.

The filamentous fungi aspergilli produce an NADP�-specificGDH involved in ammonium assimilation (24, 25). PurifiedGDH from Aspergillus niger (AnGDH) (26) exhibits sigmoidsaturation with �-ketoglutarate (K0.5 � 4.78 mM) and is com-petitively inhibited by isophthalate (Ki � 6.9 �M). AnGDHshows a distinctive feature of unidirectional inhibition of for-ward activity by 2-hydroxyethyl disulfide (2-HED) modificationwithout affecting its reverse activity (27). Surprisingly, theNADP�-dependent GDH from Aspergillus terreus (AtGDH)shows hyperbolic saturation with �-ketoglutarate (Km � 6.0mM), and both its forward and reverse activities remain unal-tered in buffers containing 2-HED. Both AnGDH and AtGDHare expressed as polypeptide with 460 amino acids, share 88%sequence identity (Fig. S1), and are active as hexamers (27).Despite their high sequence identity, AnGDH and AtGDHexhibit exceptional differences in kinetic properties; hence, astructural justification is desired.

We report the first high-resolution crystal structures of fun-gal GDH. The structures of AnGDH were solved as apoenzyme,catalytically competent ternary complexes, as well as com-plexes with reaction intermediates and an inhibitor. The struc-tures of forward-inhibited AnGDH (fiAnGDH) have also beendetermined as apoenzyme and complexed with �-ketoglutarate(AKG). Analysis of structures complemented with functionalcharacterization demonstrates the structural basis of the coen-zyme specificity and kinetic cooperativity of this enzyme. Ourdata provide direct proof for some of the reaction intermediatesof the catalytic mechanism. The results presented here arebroadly applicable to all GDHs studied so far, and some aspectsextend to dehydrogenases in general.

Results

Structural fold of AnGDH

We determined the first crystal structures of GDH fromthe fungus kingdom (Fig. 1). The structures of AnGDH weresolved as apoenzyme as well as its complex with substrate(�-ketoglutarate)– coenzyme (NADPH), reaction intermedi-ates (�-iminoglutarate (AIG)–2-amino-2-hydroxyglutarate(AHG)), the fiAnGDH–AKG complex, and inhibitor (isophtha-late) at resolutions of 2.8, 1.8, 1.75, 2.25, and 1.9 Å, respectively(Table 1). The hexamer of fiAnGDH has three subunits com-plexed with �-ketoglutarate and the rest of the subunits inan unliganded form. All the structures are of high quality asreflected by their low R-factors and good stereochemicalparameters (Table 1). Only the apo–AnGDH structure has rel-atively high R-factors, mainly due to the lower resolution andpoor redundancy of the diffraction data. All the bound ligandsare unambiguously defined in the active site of AnGDH by clear

electron densities. AnGDH has high sequence identity (Fig. S2)with other NADP�/NAD�-specific GDHs and a few extraamino acid insertions. The overall structural fold of AnGDH issimilar to the previously determined structures of Escherichiacoli GDH (EcGDH) and other GDHs (7, 12, 21–23). Each sub-unit of AnGDH consists of two domains separated by a deepcleft (Fig. 1, a and b). Domain I consists of residues 1–190 and437– 460, and domain II consists of residues 191– 436. Thesedomains are mainly composed of �-helices and �-sheets, whichare numbered as H1–H16 and �1–�13, respectively. Domain Iplays a significant role in subunit assembly and combinesprotomers leading to formation of a hexamer with 32 symmetry(Fig. 1c). Domain I is mainly engaged in binding of �-ketogl-utarate, whereas domain II facilitates binding of NADP(H).Domain II has seven �-helices and seven �-strands folded ina modified Rossmann fold (28, 29) with �7H8�8�9H9 and�11H12�12H13�13H7 as first and second motifs, respectively.These two motifs are connected by H10H11�10. Analysis of thedomain II of AnGDH–AKG–NADPH complex shows that theresidues present in the loops “�8–loop–�9” and “H9–loop–H10” are responsible for NADP(H) recognition (discussedbelow). The interface between the two domains is formed bythe interactions provided by long helices (H15 and H16). Likeother GDHs, AnGDH forms a hexameric structure that maybe considered to be composed of two trimers or three dimers.The dimeric interface is mainly formed by �1, �2, H1, and H16 ofthe substrate-binding domain I, whereas the trimeric interfaceformation is mediated by the residues located at the N terminusof H14 and the C terminus of H15. Similar interfaces have alsobeen reported for hexameric structures of EcGDH and Plasmo-dium falciparum GDH (PfGDH) (9).

Conformational flexibility in AnGDH structure

The apo–AnGDH and fiAnGDH–AKG complexes werecrystallized with a hexamer in the asymmetric unit (Fig.1c). Structural superpositions of both (apo–AnGDH andfiAnGDH–AKG) show lower root mean square deviation(r.m.s.d.) values (0.6 Å) for equivalent monomers, indicatingalmost identical conformation of these two structures. AnGDHhexamer has a cylindrical shape with approximate height anddiameter of 110 and 96 Å, respectively. Interestingly, each sub-unit of AnGDH hexamer has a different conformation (Fig. 1d).Overall structural superpositions of the A subunit with the B–Fsubunits of the AnGDH hexamer produced r.m.s.d. values of0.8, 0.4, 2.2, 2.1, and 2.2 Å, respectively. Superposition of the Dsubunit on E and F produced r.m.s.d. values of 0.3 and 0.4 Å,respectively. Furthermore, conformational differences amongthe subunits of the hexamer were also analyzed using the open-ing of the substrate/coenzyme binding cleft by measuring thedistance (Table 2 and Fig. 1e) between the C� atoms of Lys-122and Arg-280. These results indicate that three protomers at oneside of the hexamer are in an open conformation, and threesubunits at the opposite side are in a closed one. Such symmet-ric opening and closing of trimers in a hexameric assembly of aGDH were observed for the first time. Interestingly, the closedsubunits of apo–AnGDH and only an �-ketoglutarate– boundform of fiAnGDH have identical cleft opening, indicating thatthe subunits in the AnGDH hexamer may remain in both open

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Figure 1. Structural fold and flexibility of AnGDH. a, tertiary structure of AnGDH is shown as a cartoon. The helices (cylinders) are marked with H, and thestrands (arrows) are marked with �. The star at the center of two domains represents the substrate-binding site. b, topology diagram showing the arrangementof secondary structural elements. c, hexameric assembly shown in cartoon representation with each subunit with a different color. The structure is viewed froma direction perpendicular to the 3-fold axis (arrow). d, conformational variabilities of domain II among the subunits in AnGDH hexamer. e, superposition of open(purple) and super-closed (cyan) conformations of AnGDH structures showing the domain closure and structural flexibility. Bound �-ketoglutarate and NADPHare shown as sticks. The positions of the C�-atoms of Lys-122 and Arg-280 are shown as spheres. f, schematic diagram depicting conformational change inAnGDH structure upon substrate and coenzyme binding.

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(three) and closed (three) states in the absence of any ligand.The opening of the binding cleft in AnGDH–AKG–NADPHcomplex is much smaller, with a distance of 6.0 Å (Table 2 andFig. 1, e and f), and similar values were also observed for otherAnGDH ternary complexes reported in this study. Previously,the ternary complex structures of bovine GDH (21), Burkhold-eria thailandensis (BtGDH), and C. glutamicum (CgGDH) (30)have been reported with the opening cleft distances of 11.4, 9.9,and 9.4 Å, respectively. Hence, AnGDH ternary complex struc-ture is the first representative with super-closed conformation

(Table 2). The superposition of domain I (residues 2–190),domain II (residues 192–375), and hinge helix regions (residues379 – 439) were performed separately and resulted in ther.m.s.d. values of 0.4, 0.3, and 1.0 Å, respectively. These valuesindicate very little structural modulation in the two domains;however, the hinge region undergoes a substantial conforma-tional change. Although most of the residue positions remainunaltered, the side chains of Gln-12, Lys-122, Arg-193, Arg-280, Arg-407, and Arg-418 adopt different conformations inthe open and closed states of the AnGDH structures. The inter-

Table 1Data collection and refinement statisticsThe following abbreviations are used: apo-AnGDH, AnGDH structure without any ligand in the active site; AnGDH-AKG-NADPH: AnGDH structure complexed withAKG and NADPH; fiAnGDH-AKG, forward-inhibited AnGDH structure complexed with AKG; AnGDH-AIG-NADP�, AnGDH structure complexed with reactionintermediates AIG/AHG and NADP�; AnGDH-IPT-NADPH, AnGDH structure complexed with inhibitor IPT and NADPH.

Apo-AnGDH AnGDH-AKG-NADPH fiAnGDH-AKG AnGDH- AIG -NADP� AnGDH-IPT- NADPH

Data collection statisticsa

Space group P1 H32 P1 H32 H32Unit cell dimensions

a, b, c (Å) 92.8, 92.8, 111.7 174.5, 174.5, 240.4 92.3, 92.2, 111.1 173.7, 173.7, 241.9 173.5, 173.5, 241.1�, �, � (°) 103.5, 94.2, 120.1 90.0, 90.0, 120.0 103.4, 93.5, 120.4 90.0, 90.0, 120.0 90.0, 90.0, 120.0

Resolution (Å) 40.0–2.8 (2.9–2.8) 35.0–1.8 (1.9–1.8) 35.0–2.25 (2.35–2.25) 35.0–1.75 (1.85–1.75) 70.0–1.9 (2.0–1.9)Wavelength (Å) 1.5418 0.9763 1.5418 0.95372 0.9763Temperature (K) 100 100 100 100 100Observed reflections 128,417 (10,559) 1,571,927 (207,073) 270,254 (31,489) 1,103,599 (168,659) 809,288 (106,698)Unique reflections 69,072 (6502) 128,967 (18,853) 135,737 (15,991) 138,857 (21,413) 109,208 (15,446)Completeness (%) 92.0 (86.0) 99.6 (97.9) 95.5 (92.0) 99.0 (99.9) 99.9 (98.8)Rmerge (%) 6.2 (28.6) 9.2 (41.5) 9.5 (39.8) 10.9 (110.5) 5.4 (41.9)Rmeas (%) 8.8 (40.5) 9.6 (43.5) 13.4 (56.3) 11.6 (118.3) 5.8 (45.2)I/�I 11.2 (3.6) 17.1 (5.1) 8.0 (2.1) 13.4 (1.9) 24.3 (4.5)CC1/2 (%) 99.0 (83.7) 99.8 (96.4) 98.9 (67.9) 99.8 (69.9) 99.9 (61.3)Redundancy 2.0 (1.6) 12.2 (10.9) 2.0 (1.9) 7.9 (7.9) 7.4 (6.9)

RefinementResolution (Å) 35.0–2.8 34.0–1.8 33.0–2.25 34–1.75 34–1.9No. of reflections

(working set/test set)65,578/3451 1225,17/6448 128,936/6786 131,488/6920 103,732/5460

Rfactor (%) 22.6 13.5 15.8 15.5 16.8Rfree (%) 31.0 14.8 19.8 16.9 18.0No. of atoms

Protein 20,940 3669 20,933 3544 3513Water 601 674 1515 528 308AKG 0 10 30 0 0NADPH 0 48 0 0 48NADP� 0 0 0 48 0�-Mercaptoethanol (BME) 0 0 24 0 0IPT 0 0 0 0 12AIG 0 0 0 10 0AHG 0 0 0 11 0

Average isotropic B-factor(Å2) for active-site ligands

AKG 26.8 48.6NADPH 24.0 26.9NADP� 22.4IPT 20.4AIG 20.1AHG 31.6Average isotropic B-factorAverage isotropic(Å2) of all atoms

27.5 29.3 25.9 28.3 31.0

Occupancy for active-site ligandsAKG 1.0 1.0NADPH 1.0 1.0NADP� 1.0IPT 1.0AIG 0.8AHG 0.2 (0.6 for O

atom of 2-OH )r.m.s.d.

Bond length (Å) 0.011 0.014 0.011 0.012 0.011Bond angle (°) 1.42 1.65 1.43 1.65 1.44

Protein geometryRamachandran plot favored (%) 92.92 97.07 96.20 96.34 96.41Ramachandran plot allowed (%) 6.09 2.93 3.39 3.43 3.14Ramachandran plot outliers (%) 1.00 0.0 0.41 0.23 0.45

PBD codes 5XVI 5XVX 5XVV 5XWC 5XW0a Values in parentheses correspond to highest resolution shell.

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subunit salt-bridge interactions (Arg-407–Glu-403 and Lys-171–Asp-458) present between the open subunits within thehexamer are lost due to the domain closure. The domain move-ment in AnGDH analyzed by the DynDom (31) web server indi-cates symmetric domain closure of 34° and rotation of domainII by 20° due to the conformational change in the hinge helices(H14 and H15) (Fig. 1f). The conformational flexibility ofAnGDH structure plays an important role in the inter-subunitcommunication to control the substrate/coenzyme binding aswell as the homotropic cooperative interactions with its sub-strate �-ketoglutarate (discussed below).

Active site of AnGDH complexed with �-ketoglutarate andNADPH

The structure of the ternary complex of AnGDH with �-ke-toglutarate and NADPH has been determined at 1.8 Å; the elec-tron density for the substrate and coenzyme in the active sitewas unambiguous (Fig. 2a). This complex represents the firstnon-mammalian GDH showing correct orientation of theadenosine 2�-phosphate group of NADPH in the catalyticallycompetent enzyme active site. The domain closure facilitatesappropriate positioning of NADPH and �-ketoglutarate as wellas interactions of the ligands with active-site residues. �-Keto-glutarate is bound via several polar interactions involving resi-dues Lys-78, Gln-99, Lys-102, Lys-114, Asp-154, Arg-193, andAsn-346 (Fig. 2b and Fig. S3a). The distance between the re-active carbonyl carbon (C2) of �-ketoglutarate and hydridedonating/accepting carbon (C4) of the nicotinamide group ofthe coenzyme in AnGDH is 2.8 Å, and it is 3.8, 4.11, and 4.14 Å,respectively, in the bovine GDH, BtGDH, and CgGDH. InAnGDH, a water molecule located close to the �-carbon atomof �-ketoglutarate forms a short hydrogen bond (1.9 Å) with theside chain of Lys-114, implying its importance in catalysis. Thecoenzyme is held in the active-site cleft via interactions withthe residues primarily from domain II and a few others fromdomain I. The adenine ring is anchored inside a pocket formedby His-84, Ile-155, and Thr-321 (Fig. 2c and Fig. S3b). Theribose of adenosine is placed in a groove formed by the sidechains of Ser-229, Asp-252, and Ala-320. The residues Gly-228 –Ala-233 forming the GXGXX(G/A) motif (32) providehydrogen-bonding interactions to the coenzyme. Ser-229, apart of this motif, forms a hydrogen bond with the 3�-hydroxyl

group of adenosine. To position the 2�-phosphate group ofNADPH, the Asp-252 carboxylate group is pointing away fromthe ribose sugar and forms a salt bridge with the side chain ofLys-277. In NAD�-dependent dehydrogenases, the position ofAsp-252 is generally occupied by an Asp or Glu located at the Cterminus of the second �-strand of the ���-fold; these residuesform important hydrogen bonds with the 2�-hydroxyl group ofadenosine (33, 34). In AnGDH, the 2�-phosphate group isanchored primarily via direct hydrogen bonds with the sidechains of Ser-253 and Gln-282 of domain II and Lys-122 ofdomain I. Lys-277 and His-84 side chains also form water-me-diated hydrogen-bonding interactions with the 2�-phosphategroup. Alanine mutants of these five residues were generated,and the observed deviation of the measured kinetic parameters(Table 3 and Fig. S4) of the mutants confirms involvement ofthese residues in NADPH binding. The alanine mutants ofSer-253, Lys-277, and Gln-282 lose significant amount of theNADPH-dependent enzymatic activity as compared with thenative enzyme. Notably, none of these mutants as well asthe WT enzyme show any measurable NADH-dependent activ-ity. Because of the disruption of the polar interactions by ala-nine mutation of the polar residues, the apparent Km values forNADPH binding are increased to varying extents in the singlemutants. The highest Km value was observed for the K277Amutant, which also showed weak positive cooperativity towardNADPH saturation. A significant decrease in kcat is observedfor S253A and Q282A mutants with latter having the lowestvalue. The catalytic efficiency (kcat/Km) of S253A, K277A, andQ282A has decreased drastically (220, 40, and 3300 times,respectively) as compared with the WT enzyme. The catalyticefficiency lost in H84A and K122A mutants was 1.7- and 3.0-fold, respectively, and is not that significant. The measuredkinetic parameters of the NADPH-dependent activities indi-cate primary involvement of Ser-253, Lys-277, and Gln-282 inbinding the 2�-phosphate group of the cofactor.

The pyrophosphate group of NADPH is hydrogen-bonded tothe main chain of the AnGDH GXGXX(G/A) motif (formed byresidues Gly-228 –Ala-233 of domain II) and is also hydrogen-bonded with well-defined water molecules nearby. The 2�-hy-droxyl group of the ribose sugar has hydrogen-bonding inter-actions with the side chains of Arg-82, Asp-154, and Asn-346

Table 2Active-site cleft opening in different AnGDH structures

Structure ChainDistances (Å) between C�

atoms of Arg-280 and Lys-122 Position of subunits

Apo-AnGDH (hexamer) A 13.0 Upper part of hexamerB 15.1C 13.4D 19.7 Lower part of hexamerE 21.3F 19.4

AnGDH ternary complex (monomer) A 6.0fiAnGDH-AKG complex (hexamer) A 12.2 Upper part of hexamer

B 14.8C 13.3D 20.2 Lower part of hexamerE 21.3F 19.2

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(Fig. 2c). A well-defined electron density (Fig. 2a, inset) indi-cates that the C4 atom of the nicotinamide ring has tetrahedralgeometry, implying the presence of a reduced form of coen-

zyme in the enzyme active site. The nitrogen atom of the amidegroup is hydrogen-bonded to one of the oxygen atoms of theNADPH pyrophosphate moiety and the side chain of Asn-231

Figure 2. Active site of AnGDH complexed with AKG and NADPH. a, �-A weighted Fo � Fc omit electron density maps of AKG (light brown carbon) andNADPH (yellow carbon) contoured at the 3.0 � level, with the final refined models superimposed. Inset shows the tetrahedral geometry of the C4 carbon atomin the nicotinamide ring of NADPH and its close proximity for hydride transfer to the �-carbon of AKG. Covalent structures of AKG and reduced form ofnicotinamide ring are also shown. b, binding pocket for AKG in the active site of AnGDH. The residues are shown as light gray–colored carbon. Polarinteractions are shown as dotted lines. Water molecules are shown as spheres. c, NADPH-binding pocket in the AnGDH active site. Representation styleis same as in b.

Table 3Kinetic parameters for the NADPH saturation of the wildtype AnGDH and its various mutantsThe units of Km for NADPH and kcat are �M and s�1, respectively.

Wildtype H84A K122A S253A K277A Q282A

NADPHKm 24 � 2.1 25.6 � 2.2 53.8 � 0.9 44.8 � 0.7 132.4 � 1.2a 66 � 1.3kcat 198.3 � 1.3 123.7 � 0.8 145.6 � 0.4 1.7 � 0.3 28.3 � 0.8 0.17 � 0.03kcat/Km 8.3 � 0.8 4.8 � 0.4 2.7 � 0.05 3.7 � 10�2 � 0.005 2.1 � 10�1 � 0.005 2.5 � 10�3 � 4 � 10�4

a The S0.5 value is reported. The data are represented with the standard error of the mean.

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(Fig. 2c). The distance between the �-carbonyl carbon (C2) of�-ketoglutarate and the C4 atom of nicotinamide ring is 2.8 Å,indicating that reactive states of the substrate and coenzyme aretrapped in the enzyme active site. Such a close interactionbetween the coenzyme and substrate has never been capturedin crystal structures of GDHs reported previously.

Reaction intermediates in the AnGDH active site

The enzymatic reaction was carried out during the crystalli-zation process following the scheme (Fig. 3a) in the presence ofNADP� so that �-iminoglutarate (AIG) formed in the enzymeactive site does not get reduced to form L-glutamate. The highresolution (1.75 Å) electron density map (Fig. 3, b, panel i, and c)shows the presence of reaction intermediates and NADP� inthe active site. Initially, NADP� and �-iminoglutarate wererefined (Fig. 3b, panel ii) in the active site. However, a positiveresidual Fo � Fc electron density (Fig. 3b, panel ii) remainedconnected with �-iminoglutarate. Refinement of 2-amino-2-hydroxyglutarate with partial occupancy could satisfy theremaining positive electron density (Fig. 3b, panel iii).

This complex presents the first structural proof of formationof the �-iminoglutarate and 2-amino-2-hydroxyglutarate asintermediates during the reaction catalyzed by a GDH. Notably,the binding mode of �-iminoglutarate and �-ketoglutarate is

identical. The �-imino group has polar interactions with thecarboxylate group of Asp-154 and the main chain carbonylgroup of Gly-153 (Fig. 3d and Fig. S5b). The �-hydroxyl groupof 2-amino-2-hydroxyglutarate is hydrogen-bonded to the Lys-114 side chain (Fig. 3d and Fig. S5a). The carbonyl group ofGly-153 adopts two alternative conformations in this structure.The amino group of Lys-114 side chain acquires different con-formations in AnGDH–AKG–NADPH and AnGDH–AIG–NADP�. The distance between the �-carbon atom of �-imino-glutarate and the C4 atom of the nicotinamide moiety ofNADP� in AnGDH–AIG–NADP� structure is 3.0 Å. Thebinding mode of NADP� in the AnGDH–AIG–NADP� com-plex is almost identical as that observed for NADPH in theAnGDH–AKG–NADPH complex. The only difference is thatin the �-iminoglutarate-bound structure the 2�-phosphategroup has moved closer to Lys-277, and the side chain of thisresidue directly interacts with the phosphate oxygen atom(Fig. S5c). These structural data support the accommodationof the tetrahedral intermediate for the first time, an entitypostulated before but with no such direct structural evi-dence. Our data directly implicate the formation of an �-imi-noglutarate and 2-amino-2-hydroxyglutarate as the reactionintermediates in the catalytic mechanism of glutamate dehy-

Figure 3. Active site of AnGDH complexed with AIG, AHG, and NADP�. a, reaction steps in the AnGDH active site leading to the formation of AHG and AIG.b, electron density map guided the refinement process of the reaction intermediates in the active site. (i) Initial �-A weighted Fo � Fc omit electron density map(green) contoured at the 3.0 � level showing predominant features of AIG and NADP�. (ii) Positive Fo � Fc omit map (green) contoured at the 3.0 � level indicatespartial occupancy of AHG. (iii) Refined 2Fo � Fc map (blue) contoured at the 1.0 � level, after refinement of AIG, AHG, and NADP�. c, �-A weighted Fo � Fc omitelectron density maps contoured at the 3.0 � level, with the final refined models AIG (light-magenta carbon), AHG (light-brown carbon), and NADP� (graycarbon) superimposed. d, binding pocket for AIG and AHG in the active site of AnGDH. The residues are shown as light-gray– colored carbon. Polar interactionsare shown as dotted lines.

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drogenase, as invoked previously using spectroscopic analy-sis (13, 17, 19, 20).

Binding mode of an inhibitor isophthalate in the AnGDHactive site

This is the first structure of an isophthalate (IPT)-boundGDH (Fig. 4a). In fact, there is no isophthalate complexed pro-tein structure available in the PDB. Isophthalate occupiesthe same binding pocket where �-ketoglutarate binds in theAnGDH active site (Fig. 4b and Fig. S6). All eight carbons ofisophthalate are present in the plane of the benzene ring, butthe oxygen atoms of the carboxylate groups are out of thisplane. In contrast, the crystalline form of free isophthalate isreported to form a planar structure with all the atoms residingin one plane (35). The two carboxylates of isophthalate makeidentical interactions as those observed for �-ketoglutarate.The active site-bound NADPH has an identical conformationin both AnGDH–AKG–NADPH and AnGDH–IPT–NADPHcomplexes. Although the two carboxylate groups of the sub-strate and the inhibitor occupy conformationally similar posi-tions, their other carbon atoms do not. Interestingly, due to thepresence of a hydrophobic inhibitor, the water moleculeobserved close to Lys-114 in the AnGDH–AKG–NADPH isdisplaced in the AnGDH–IPT–NADPH complex. Notably,isophthalate has caused �58.9° rotation of the peptide bondbetween Gly-153 and Asp-154 as compared with the �-ketogl-utarate-bound structure (Fig. 4b). The conformational changeof Gly-153 main-chain carbonyl group is enough to positionthe aromatic ring of isophthalate in the binding pocket, sug-gesting the plasticity of the AnGDH active site. This plastic-ity may be essential for binding the reaction intermediatesduring catalysis.

Hexameric structure of fiAnGDH with open and partiallyclosed subunits

In the hexameric fiAnGDH structure, three monomers (ofone trimer) are in partially closed conformation and complexedwith �-ketoglutarate. The remaining unliganded three subunits(of other trimer) have open conformations (Table 2). The cova-lent modification of Cys-141 in all six subunits is clearly visiblein the electron density map (Fig. S7). The �-ketoglutarate mol-ecules bound in the active site of partially closed fiAnGDH sub-

units have different conformations when compared with thoseobserved in AnGDH–AKG–NADPH complex (Fig. 5, a and b).The striking conformational difference is seen for the �-car-bonyl group, which mainly forms hydrogen bonds with the sidechain of Lys-114 and main chain –NH group of Gly-80 infiAnGDH–AKG complex, whereas in AnGDH–AKG–NADPHcomplex, this group is primarily interacting with the side chainof Asp-154 as well as the main chain carbonyl group of Gly-153.The carboxylate group of Asp-154 interacts with the side chainsof Arg-82. The spatial arrangement of the AnGDH active sitesuggests that Arg-82 might play a crucial role in maintainingthe ionization state of Asp-154. Arg-82 is highly conservedamong NAD�- and NADP�-dependent GDHs (Fig. S2). TheKm, kcat, and kcat/Km values for NH4

� binding to the R282Qmutant are 22.3 � 1.1 mM, 10.3 � 0.5 s�1, and 4.6 � 10�1 �0.02 mM�1 s�1, respectively (Fig. S8). For the native enzyme, theKm, kcat, and kcat/Km values for NH4

� are 1.4 � 0.2 mM, 106.5 �0.9 s�1, and 76.1 � 9.3 mM�1 s�1, respectively. An almost16-fold increase in Km value and a 165 times decrease in cata-lytic efficiency of R82Q mutant are consistent with the role ofArg-82 in AnGDH catalysis as proposed above.

In the AKG-bound fiAnGDH monomers, an active-sitewater molecule is visible, and it is hydrogen-bonded to the car-boxylate group of Asp-154 and the main-chain carbonyl groupof Gly-153 (Fig. 5a). This water molecule has not been observedbefore in any of the available GDH structures. Position of thiswater defines the space needed for ammonia binding during thecatalytic conversion of �-ketoglutarate to L-glutamate (see“Discussion”).

Intersubunit interactions in AnGDH structures

Kinetic measurements of AnGDH showed sigmoidal satura-tion with �-ketoglutarate and hyperbolic saturation withNADPH (Fig. 6, a and b). We performed careful analysis of thehexameric structures of this enzyme to decipher the structuralbasis of this allosteric feature. It is evident (Table 2) that indi-vidual subunits undergo conformational changes in the pres-ence and absence of substrate, coenzyme, and ligand. Duringthe catalytic cycle, domain I of each subunit of the hexamericassembly remains almost unchanged. However, domain IImoves closer to domain I, and it undergoes clockwise rotation(around 20°) with respect to the 3-fold axis of the hexamer. The

Figure 4. Active site of AnGDH complexed with IPT and NADPH. a, �-A weighted Fo � Fc omit electron density maps of IPT (gray carbon) and NADPH (yellowcarbon) contoured at the 3.0 � level, with the final refined models superimposed. b, comparison of mode of binding of AKG (cyan) and isophthalate (green).

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structural arrangement suggests that only domain II facilitatesinteractions with the neighboring hexamers. Because of closureand rotation of domain II, the inter-subunit interactions aredifferent in closed subunits as compared with the open ones.

Each subunit of the central hexamer has 3-fold symmetry-related lateral interactions with two neighboring subunits fromdifferent hexamers present in the same horizontal plane (Fig. 6,

c– e). On one side (with the A–C subunits) of the hexamer, thelateral interactions are mediated by the side chains of Asn-335and the main chain carbonyl group of Thr-362. On the otherside of the hexamer (with D–F subunits), the H13 helix (residues351–363) of one monomer is packed in the groove formed bythe H10 helix (residues 283–287) and �10 (residues 298 –302)from a subunit of the neighboring hexamer through hydropho-

Figure 5. Conformational flexibility of AKG in the active site of AnGDH. a, binding mode of AKG (light brown carbon) in the active site of fiAnGDH (graycarbon). The �-A weighted Fo � Fc omit electron density map contoured at the 3.0 � level is also shown as green mesh around the final refined model of AKG,and the water molecule is presented as a sphere. b, conformational differences of AKG in the coenzyme-bound ternary (blue carbon) and unbound binary(brown carbon) AnGDH complexes.

Figure 6. Interactions among the hexamers of fiAnGDH–AKG complex in the crystal. a, AKG binding to AnGDH (3 �M) active site shows cooperativity. b,NADPH binding to AnGDH (6 �M). Insets in a and b show the change in fluorescence intensities (colored lines) upon addition of substrate (AKG) and coenzyme(NADPH), respectively, to the AnGDH in one of the experiments. c, interactions among the hexamers of the fiAnGDH–AKG complex in the crystal. Each subunitof the hexamers is shown in a different color (green; cyan; yellow; magenta; gray; and blue). The lateral and vertical arrangements of the hexamers in the crystalare shown. d, symmetric lateral organization of the hexamers in the crystal, viewed from the top. e, zoomed in view of the lateral interactions present in one sideof the hexamer. f, vertical arrangement of the hexamers. g, representation of the interactions present in the vertical orientation.

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bic and van der Waals interactions. The methyl groups of Thr-356 and Thr-362 side chains from one subunit have hydropho-bic contacts with the side chains of Ala-302 and C� carbonatom of Ser-285, respectively, of the other subunit. Thereforethe lateral interactions are mediated by Ser-285, Ala-302, Asn-335, Thr-356, and Thr-362.

The inter-subunit interactions between the hexamers in thevertical directions are asymmetric (Fig. 6, c, f, and g). Most of thecontacts are mediated by the residues from domain II of A sub-unit from one hexamer as well as D and E subunits of two otherhexamers. The side chains of Glu-217 of the A subunit of thecentral (first) hexamer form a salt-bridge interaction with theside chain of Lys-299 of the E subunit of the second hexamer.The side chains of Lys-339 of the A subunit also form hydrogen-bonding interaction with the side chain of Asn-296 of the Esubunits of the second hexamer. Gln-216 of the A subunitmakes hydrogen-bonding interactions with the side chain ofLys-299 and the main chain of Ile-259 and Val-310 from the Esubunit. The loop region containing residues Ile-259 –Gly-263of the E subunit of the second hexamer is also in close proximityof Thr-213 from H7 of subunit A of the central hexamer. On theother side of domain II of the A subunit, Lys-297 forms salt-bridge interactions with Glu-262 from the D subunit of thethird hexamer. Ser-246 side chain of A subunit also forms ahydrogen bond with the side chain of Lys-312 from the D sub-unit of the third hexamer. Domain II of the B subunit of thecentral hexamer has only two polar interactions mediated bythe side chains of Gln-216 and Ser-218 with the side chains ofAsn-214 and Gln-216, respectively, from the F subunit of thefourth hexamer. Domain II of the C subunit of the central hex-amer does not have any interaction in the vertical direction withother hexamers. The difference in the extent of interactionswith monomers in the vertical direction is mainly due to theclosure and rotation of domain II. Thus, the residues primarilyinvolved in vertical contacts are Gln-216, Glu-217, Ser-246,Glu-262, Asn-296, Lys-299, and Lys-312.

Analysis of the crystal packing of the AnGDH ternary com-plex (AnGDH–AKG–NADPH) revealed that the crystallo-graphichexamersarecomposedofsymmetricallyidenticalmono-mers arranged in the crystal with a large (104 Å diameter)solvent cavity (Fig. S9). The ternary complexes in the crystallo-graphic hexamer are in the super-closed conformations (Table2)andhaveonlydiagonalinter-subunitinteractions.Eachmono-mer of a hexamer is interacting with a monomer from the otherhexamer by salt-bridge interactions mediated by the side chainof Lys-299 and Glu-262. However, two inter-subunit salt-bridge interactions (Arg-407–Glu-403 and Lys-171–Asp-458)between the open subunits within a hexamer are lost because ofthe domain closure. Formation and disruption of several inter-actions among the subunits might play a crucial role toward theallosteric property of AnGDH.

Discussion

GDH is an essential enzyme in all living organisms. Despiteextensive studies (17, 30) in the last 40 years on prokaryotic aswell as eukaryotic GDHs, the structural basis of the coenzymespecificity and the mechanistic features of this enzymeremained incomplete. Also, no structural information was

available for a fungal enzyme. This prompted us to performstructural studies on A. niger GDH (AnGDH). Another reasonfor this study was to understand the structural basis of thecooperative nature of this fungal enzyme.

This study presents the high resolution crystal structure ofsubstrate- and cofactor-bound GDH Michaelis–Menten com-plex. We report the first fungal GDH structure. The hexamericAnGDH is unique with three open subunits at one side andthree partially closed protomers at the other side. Such hexam-eric GDH structure has not been reported before. Partiallyclosed subunits bind the substrate �-ketoglutarate. The posi-tion of the water attached to Asp-154 and Gly-153 defines theammonium ion-binding pocket, which had remained unre-solved. The binding pocket for the 2�-phosphate group of thecoenzyme is better defined by our structural data. The structureof the AnGDH–AKG–NADPH complex provides a glimpse ofthe super-closed catalytically competent enzyme with substrateand coenzyme at a favorable distance for hydride transfer. Thestructure of AnGDH–AIG–NADP� complex captures the for-mation of �-iminoglutarate and 2-amino-2-hydroxyglutarateduring the reaction. AnGDH–IPT–NADPH complex is thefirst structure of a protein–isophthalate complex and revealsthe plasticity of the enzyme active site. Implications of the elu-cidated structures to the GDH reaction mechanism, coenzymerecognition, and substrate cooperativity are discussed below.

Structural basis of �-ketoglutarate cooperativity in AnGDH

Our structural data provide the possible explanation of the�-ketoglutarate– dependent cooperativity in AnGDH. It is evi-dent that due to the inter-subunit interactions, the protomersof a catalytically incompetent hexamer are locked either in anopen or partially closed conformation. In the super-closed con-formation, the enzyme gains a catalytically competent form.These different conformational states can be directly correlatedto the cooperative behavior of AnGDH. The Monod–Wyman–Changeux (MWC) model (36) and the Koshland-Nemethy-Filmer (KNF) (37) model are the two generally accepted modelsused to explain the kinetic cooperativity in a multimeric allos-teric enzyme. According to the MWC model, the subunits ofthe enzyme are present in a reversible equilibrium between alow-affinity tensed (T) and a high-affinity relaxed (R) state inthe absence of the ligand. When added, the ligand would bind tothe R-state, and the equilibrium would adjust by convertingmore of the T-state subunits to the R-state, thereby leading topositive cooperativity. In contrast, the KNF model indicatesthat ligand binding to one subunit can induce conformationalchanges in the other subunits resulting in an increase in ligandaffinity (and hence positive cooperativity).

Analysis of the structural data collated so far suggests that theunliganded structure of apo-AnGDH represents the low-affin-ity state (resting-state), in which half of the hexameric enzyme(one trimer) remains as open/partially closed conformation(Fig. 7). Another low-affinity form (resting-state) of the enzymewith all its six subunits in the open conformation may well exist;however, we have not encountered such a structure so far. Thehexameric unit with a trimer of three �-ketoglutarate– bound,partially closed subunits at one side and the trimer with threeopen subunits on the other side represents the T-state and is

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also not catalytically competent. Closing and opening of onetrimer subunit at a time also correlates well with the measuredHill coefficient of 2.5 for �-ketoglutarate saturation (26). Bind-ing of �-ketoglutarate and NADPH to all six subunits wouldgenerate a fully active enzyme species (R-state); this is consis-tent with the biphasic kinetic response to incubation with theproduct NADP� and higher initial velocities with prior incuba-tion of AnGDH with �-ketoglutarate and NADPH (26). There-fore, the homotropic �-ketoglutarate interactions of AnGDHmay possibly be described by the mechanism (Fig. 7) followingthe MWC model.

Our data present the first structural evidence for the allos-teric regulation in smaller GDHs. Based on the crystal struc-tures of bovine GDH, the allosteric regulation in additionalantenna domain-containing mammalian GDH has been previ-ously reported to follow the KNF model (38). The MWC allos-teric model observed in AnGDH may be applicable to smallerGDHs without the antenna domain, as this model nicely corre-lates with the measured Hill coefficient of 5.9 (39) of CsGDH.This implies that collective conformational transition of all sixsubunits in the hexamer contributes toward CsGDH positivecooperativity. The amino acid residues engaged in forminginter-subunit contacts in the AnGDH hexamer are replacedwith other residues in AtGDH (sequence alignment; Fig. S1).Such interactions may account for the absence of �-ketogl-utarate cooperativity in AtGDH. A systematic mutation analy-sis of these residues is expected to convert an allosteric AnGDHinto a Michaelian enzyme and to allow further probing into themechanistic details of the allosteric regulation in this enzyme aswell as other smaller GDHs.

Structural basis of NADP(H) recognition by AnGDH

The precisely defined position of the coenzyme in the high-resolution electron density map of our AnGDH structuresrationalizes the structural basis of coenzyme recognition in thisenzyme as well as in other NADP�-dependent GDHs. Struc-tural and mutagenesis data indicate that Lys-122, Ser-253, Lys-277, and Gln-282 are the primary determinants for coenzymebinding in AnGDH. In the AnGDH-complexed structures, theLys-277 side chain makes a direct (Fig. 8a) or water-mediated

(Fig. 2c) contact with the 2�-phosphate group of coenzyme.This observation suggests that the ionization state of the2�-phosphate group of NADP(H) might dictate its interactionswith these residues (Fig. 8, a and b).

In the NADP�-bound AnGDH–AIG–NADP� complexstructure, the 2�-phosphate group might be dibasic (Fig. 8b);conversely, in other coenzyme-bound structures this groupmight be monobasic. Lys-277 is conserved in all the NADP�-dependent GDHs (Fig. 8c). Interestingly, only K277A AnGDHmutant shows mild positive cooperativity toward NADPH,whereas the other mutants and WT enzyme show no suchkinetic behavior. This result makes Lys-277 of AnGDH aunique residue governing the binding of 2�-phosphate ofNADP(H).

One of the oxygen atoms (O2) of the 2�-phosphate groupmight remain neutral in both forms due to its close proximity tothe Asp-252 side chain (Fig. 8, a and b), which is possibly neg-atively charged as it interacts with the side chains of Gln-282and Lys-277. Although in AnGDH the Asp-252 carboxylategroup does not interact directly with the phosphate group, itmight still play a crucial role in positioning Gln-282 and Lys-277 side chains (Fig. 8a). In AnGDH, Ser-253 and Gln-282 sidechains form hydrogen bonds with O2 oxygen atom due to itsclose proximity. Similar interactions of NADP� 2�-phosphategroup oxygen atom with threonine and arginine side chains arealso observed in Lactococcus lactis 6-phosphogluconate dehy-drogenase (LlPGDH) (40).

Both Asp-252 and Ser-253 of AnGDH are strictly conservedin the NADP�-dependent GDHs (41). Asp-252 is the signatureC-terminal end residue of the second �-strand of the ���-fold(Fig. 8d), and its negatively charged side chain has been so farbelieved to destabilize the binding of NADP� 2�-phosphate (42,43). Our results depict the essentiality of Asp-252 for optimalorientation of Lys-277 and Gln-282, which facilitate NADP�

binding. The presence of Lys-122, Lys-277, and Gln-282 createsa positively charged surface (Fig. S10) for binding the 2�-phos-phate of NADP(H) in AnGDH. In other NADP�-dependentGDHs, this Gln-282 is replaced by an amino acid residue with aproton donor –NH group (Fig. S2). The lowest catalytic effi-

Figure 7. Structural basis of AKG homotropic cooperativity in AnGDH. Schematic model represents the cooperativity in AnGDH.

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ciency of the AnGDH Q282A mutant points to the importanceof this residue in coenzyme recognition. In EcGDH (12),CbGDH (30), the side chain of an arginine residue, might servethe same purpose.

The other oxygen atom (O4) of the 2�-phosphate groupshould remain negatively charged and form an ionic interactionwith a suitable residue on the protein. The Lys-122 of AnGDHsatisfying this ionic interaction is highly conserved in NADP�-dependent GDHs. The similar role for an equivalent lysine res-idue in CgGDH has also been proposed (30, 44). The O3 oxygenatom of the 2�-phosphate may remain neutral or be negativelycharged. The existence of two different ionization states of the2�-phosphate group of NADP(H) was proposed before (32) butwithout any relevant structural data. The interactions presentin the NADP�- and NADPH-bound AnGDH structures pro-vide the first direct evidence justifying the proposed ionizationstates of the 2�-phosphate group. However, further high-re-solution structures, systematic mutagenesis studies, kineticparameter measurements, and quantum mechanics/molecularmechanics calculations would prove valuable.

GDH reaction mechanism: Conversion of �-ketoglutarate toL-glutamate

The conversion of �-ketoglutarate to L-glutamate by GDHtakes place in sequential steps as suggested through the exten-sive biochemical studies in last 4 decades. The identification oftwo active-site lysine residues (45–48) prompted the sugges-

tion that the reaction mechanism proceeds through formationof a Schiff’s base (49). However, this idea was ruled out due tolack of experimental evidence (50). Later, based on isotopeexchange rates and spectroscopic studies, it was proposed thatthe reductive amination of �-ketoglutarate proceeds throughan enzyme-bound �-iminoglutarate intermediate (13, 19, 50,51). The reaction mechanism involves the nucleophilic attackby ammonia on the �-ketoglutarate of the GDH–NADPH–�-ketoglutarate ternary complex. The �-ketoglutarate carbonyloxygen was proposed to be protonated by a catalytic lysineforming the carbinolamine intermediate. Elimination of waterfrom this carbinolamine generates the �-iminoglutarate inter-mediate, which was spectroscopically identified in bovine GDH(17). Proton transfers in this step are possibly assisted by a car-boxylate group of an aspartate (Asp-165 in CsGDH) (13). Thereduced coenzyme, stacking in close proximity to the newlyformed imino group, completes the facile hydride transfer toform L-glutamate. Besides a catalytic carboxylate-containingresidue, two active-site lysines (Lys-113 and Lys-125 inCsGDH) are also conserved in all NADP�-dependent GDHs.Despite numerous mechanistic studies, the direct structuralevidence to capture the reaction intermediates was unavailable,and the ammonia binding pocket was yet to be established.

The constellation of AnGDH active site residues and theirbinding mode with substrate, coenzyme, and reaction interme-diates unraveled the snapshots of steps that occur during the

Figure 8. Structural basis of cofactor selectivity in AnGDH. a, stereo view showing the interactions responsible for recognition of NADPH by AnGDH. Theresidues are shown in light-brown– colored carbon. Part of the bound NADPH is shown with a gray-colored carbon. The polar interactions are shown by dottedlines. The final 2Fo � Fc electron density map contoured at the 1� level is also shown as purple-colored mesh. b, schematic representation of different ionizationstates of the 2�-phosphate group of NADPH in the AnGDH active site. The red sphere on the right panel represents the oxygen atom of water. c, segment ofpolypeptide responsible for coenzyme recognition in NADP�-dependent GDHs. The GDH sequences from A. niger (An), E. coli (Ec), Agaricus bisporus (Ab),Streptococcus suis (Ss), Salmonella enteric (Se), C. glutamicum (Cg), Pseudomonas aeruginosa (Pa), Penicillium chrysogenum (Pc), Methylobacillus flagellates (Mf),Mycobacterium smegmatis (Ms), and Saccharomyces cerevisiae (Sc) have been used for the alignment. The secondary structural elements and residue numbersof AnGDH are shown at top of the alignment. Ten conserved residues in Rossmann fold essential for 2�-phosphate group recognition in NADP�-dependentGDHs are numbered at bottom of the alignment. The strictly conserved residues are shown as white on the red-colored background. d, secondary structural motif(with 10 residues as sticks) involved in NADPH recognition in AnGDH.

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catalytic reductive amination of �-ketoglutarate to L-gluta-mate. Our structural analysis reveals NADPH binding induces amajor conformational change that places the substrate andcoenzyme in a catalytically correct orientation. A water mole-cule-forming hydrogen bond with Lys-114 in AnGDH struc-tures implies its catalytic importance. The ionic interactions ofAsp-154 with Arg-82 and Lys-114 suggest that two latter resi-dues might play a crucial role in the catalysis. In a catalyticallycompetent active site, the carboxylate group of Asp-154 is likelyto remain negatively charged by interacting with the positivelycharged side chains of Arg-82 and Lys-114 (Fig. 9). Our kinetics

data on the R82Q AnGDH mutant and strict conservation ofthis residue (Fig. S2) among other GDHs implicate involvementof Arg-82 in the catalytic reaction mechanism. The importanceof Lys-114 in catalysis was demonstrated before in CgGDH(30). The Asp-154 side chain and the main chain of Gly-153might play a crucial role in anchoring an ammonium ion. Theposition of a water molecule, hydrogen-bonded to these twogroups in the AKG-bound subunits of the fiAnGDH structure(Fig. 5a), supports this view. Our structural analysis promotesthe idea that the negatively charged carboxylate group ofAsp-154 and the polarized main-chain carbonyl group of

Figure 9. Reaction mechanism for reductive amination of �-ketoglutarate by AnGDH as supported by the structural data.

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Gly-153 are involved in positioning the NH4� ion in the active

site. Reported weaker ammonium ion affinity for EcGDHD165S as compared with the WT enzyme with almostunchanged affinity for NADPH and �-ketoglutarate (52)agrees with this proposition.

Our data provide direct structural evidence supporting thefollowing AnGDH reaction mechanism (Fig. 9) similar to theschemes proposed for the GDH reaction in general. First,the Asp-154 carboxylate group deprotonates NH4

� and facili-tates nucleophilic attack by ammonia onto the �-carbon of�-ketoglutarate. The generated oxyanion becomes protonatedby the neighboring water molecule located near Lys-114; this inturn regenerates the water molecule by proton donation. A tet-rahedral intermediate (2-amino-2-hydroxyglutarate) is formedin the active site, and the AnGDH–AIG–NADP� structuredirectly supports formation of such an intermediate (Fig. 3b).Elimination of a water molecule leads to �-iminoglutarate for-mation. Subsequently, the hydride transfer from NADPH to�-iminoglutarate forms L-glutamate. The orientation of thenicotinamide ring of NADPH in the AnGDH–AKG–NADPHcomplex structure represents the catalytically competent con-formation of the coenzyme capable of hydride transfer. Theo-retical calculation on alcohol dehydrogenase reported (53) therequired distance for hydride transfer between NAD� and sub-strate to be around 2.7 Å; the corresponding distances observedin the catalytically competent AnGDH structures are consis-tent. On the whole, collated structural data amply illuminatethe key features of the GDH reaction mechanism.

Conclusions

Our results provide the structural basis of three importantaspects related to catalysis by GDH: (a) cofactor specificity, (b)allosteric regulation, and (c) reaction mechanism. We havedetermined the first crystal structures of a fungal glutamatedehydrogenase. The complexed structures of AnGDH presentdirect evidence of formation of �-iminoglutarate and 2-amino-2-hydroxyglutarate as reaction intermediates. The differentconformational states of AnGDH structures suggest that theallosteric regulation in this enzyme follows the MWC model.The structural data reveal that the 2�-phosphate group ofNADP(H) anchored to NADP(H)-GDH might have two possi-ble ionization states. These findings resonate with other dehy-drogenase mechanisms as well.

Experimental procedures

AnGDH expression and purification

The expression and purification of recombinant AnGDHwere performed as described previously (5, 26, 27), with minormodifications. A single colony of E. coli BL21 (DE3) with theAnGDH expression construct was grown overnight at 37 °C inLB broth medium with ampicillin (100 �g/ml). The culture (1%v/v) was re-inoculated into the LB medium containing ampicil-lin (100 �g/ml) and grown at 37 °C until an optical density of 0.5at 600 nm, and then protein expression was induced by additionof isopropyl 1-thio-�-D-galactopyranoside (400 �M). Proteinexpression was done by growing the culture at 22 °C for 12 h.The cell pellet obtained from 1 liter of culture was suspended ina lysis buffer (Buffer A: 100 mM potassium phosphate buffer, pH

7.5, 1 mM EDTA, and 1� protease inhibitor mixture). After celldisruption using ultrasonication, the cell-free extract was pre-pared by centrifugation at 12,000 rpm, and the supernatant wascollected. Ammonium sulfate saturation was performed, andthe pellet obtained after 30 –70% saturation was dissolved inBuffer B (20 mM potassium phosphate buffer, pH 7.5, and 1 mM

EDTA). The sample was further desalted using HiPrep 26/10desalting column. Next, the protein sample was loaded onto a50-ml CR-12 dye affinity column (Novacron Red LS-BL cou-pled through an epoxy spacer arm to Sepharose), and elutionwas performed by a linear gradient of potassium chloride. Theeluted sample was desalted and loaded onto a DEAE-Sepharose(5 ml, HiTrap) column, and bound proteins were eluted by alinear gradient of potassium chloride. The final purification ofAnGDH was performed using a Superdex 200 16/60 gel-filtra-tion column. The purity of the protein was analyzed on SDS-PAGE (54). Protein concentrations were measured using Brad-ford’s method (55).

Enzyme assay

The enzyme activity of pure AnGDH was assayed asdescribed previously (5). Reductive amination of �-ketogl-utarate to L-glutamate is accompanied by oxidation of NADPHto NADP�. Therefore, the enzyme activity was measured by theinitial rate of disappearance of NADPH absorbance at 340 nm.The change in absorbance (A340) was recorded per min.Forward reaction was carried out in 1 ml of total reactionmixture containing 100 mM Tris buffer, pH 8.0, 10 mM �-ketoglutarate, 10 mM ammonium chloride, and 0.1 mM

NADPH. One enzyme activity unit refers to the amount ofenzyme required to oxidize 1 �mol of NADPH per minunder the standard assay conditions.

Site-directed mutagenesis

PCR-based site-directed mutagenesis was performed to gen-erate Ala-substituted AnGDH mutants at residues His-84, Lys-122, Ser-253, Lys-277, and Gln-282 using the pET43.1bAnGDHexpression vector. R82Q mutant was prepared in a similarway. The primers used for generating these mutants arelisted in Table S1. Mutations were confirmed with DNAsequencing, and the expression constructs of these mutantswere transformed into GDH E. coli BL21 (DE3) (56).Expression, purification, and activity assays of the mutantenzymes were performed following the same procedures asdescribed for recombinant WT AnGDH. Correctness offolding of the mutants was confirmed by circular dichroism(CD) measurements.

Kinetics of WT and mutant AnGDH

NADPH saturation of the WT AnGDH and its mutant forms(H84A, K122A, S253A, K277A, and Q282A) was performedusing reductive amination assay. This reaction was followed atpH 8.0 with varying substrate (NADPH) concentrations. Thestandard assay (as mentioned under “Enzyme assay”) was suit-ably modified. The substrate conversion was maintained below10% to achieve initial velocity. The NADPH concentration wasvaried, and the enzyme and other substrate concentrations

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were kept constant. The saturation of NADH was performed inparallel.

The assay was suitably modified for the ammonia saturationkinetics of WT AnGDH and its R82Q mutant. NADPH and AKGconcentrations were kept fixed, and ammonium chloride concen-tration was varied with a suitable amount of the enzyme. All theseexperiments were performed in triplicate at room temperature.

Fluorescence quenching experiment

Purified AnGDH (1 mg/ml) in 20 mM phosphate buffer, pH7.5, was used for measuring the substrate- and cofactor-in-duced fluorescence intensity change. All the experiments wereperformed in a fluorescence spectrophotometer (JASCO). Forthe tryptophan quenching in AnGDH, the excitation wave-length of 295 nm was used, and the emission wavelength was inthe range of 300 –500 nm. The excitation and emission bandwidths were kept at 2.5 nm, and slit width used was 2.5 nm. Thereaction mixtures contained a fixed concentration (3 �M) ofAnGDH and variable concentrations of �-ketoglutarate in atotal reaction volume of 0.5 ml assembled in a quartz cuvette,and a path length of 10 mm was used. Different concentrationsof �-ketoglutarate used for the measurements were 2, 5, 7, 9, 15,and 20 mM. In separate experiment, a fixed concentration (6�M) of AnGDH was used with NADPH of varying concentra-tions (0.2, 0.5, 1, 2, 4, 6, 8, 12, 15, and 18 �M). The observedfluorescence intensities were corrected for the inner filter effectin the experiments. All the spectral measurements were carriedout at 25 °C and in triplicate.

Preparation of forward-inhibited AnGDH

The forward-inhibited AnGDH (fiAnGDH) was prepared bytreating the purified enzyme with 2 mM 2-HED and incubating themixture at 37 °C as described previously (27). Forward inhibitionof the enzyme was confirmed by activity measurements as dis-cussed above. The fiAnGDH preparation was further buffer-ex-changed with Buffer B, concentrated to 12 mg/ml, and stored at4 °C.

Crystallization

Active and forward-inhibited forms of AnGDH were crystal-lized using sitting or hanging drop vapor diffusion method at22 °C. Initial crystallization screens were set up using commer-cially available screen solutions: (a) JCSG Core-I suite (Qiagen),(b) PEGs suite (Qiagen), and (c) JCSG plus suite (MolecularDimensions), with a Phoenix (Art Robins) crystallization robotavailable at the “Protein Crystallography Facility,” Indian Insti-tute of Technology, Bombay, India. Crystallization trials forapo-AnGDH with a protein concentration of 7 mg/ml were setup using factorial 1 screening conditions (57). The first crystalsof apo-AnGDH were obtained in a condition containing 15%(w/v) PEG 3350, 0.1 M Tris-Cl, and 0.2 M NaCl within 1 week.Further optimization of this condition using several additivesproduced the best quality apo-AnGDH crystals in a motherliquor having 20% (w/v) PEG 3350, 0.1 M NaCl, 0.1 M Tris-Cl,pH 8.5, and 0.01 M BaCl2.

Crystallization of AnGDH complexed with �-ketoglutarateand NADPH (AnGDH–AKG–NADPH) was done as reportedbefore (5). For complex formation, AnGDH (12 mg/ml) was

mixed with �-ketoglutarate and NADPH for 30 min at 25 °Cachieving 0.6 mM final concentrations of the substrate andcoenzyme. The crystallization screens were set up, and the ini-tial crystals of the AnGDH–AKG–NADPH complex wereobserved within 1 week in a condition containing 0.1 M sodiumcitrate, pH 5.5, and 20% (w/v) PEG 3000. These crystals grew totheir maximum size within 1 week and were further used fordiffraction studies.

To obtain crystals of AnGDH complexed with isophthalateand NADPH (AnGDH–IPT–NADPH), the concentrated (12mg/ml) protein solution was mixed with these compounds, andthe mixture was incubated for 30 min at 25 °C. The final concen-trations of isophthalate and NADPH in the mixture were 0.8 and0.6 mM, respectively. Crystallization screens of this complex wereset up, and the best crystals were obtained in a condition contain-ing 0.1 M MES, pH 6.0, 30% (v/v) PEG 200, and 5% (w/v) PEG 3000.The crystals grew to their maximum size within 2 weeks.

Preparation of AnGDH complex with �-iminoglutarate(AnGDH–AIG–NADP�) was done by incubating the mixtureof enzyme (12 mg/ml) with �-ketoglutarate, NADP�, andammonium chloride at 25 °C for 5 min. The final mixture con-tained 1 mM �-ketoglutarate, 1 mM NADP�, and 0.5 M ammo-nium chloride. Crystallization screens of this complex were setup. After obtaining the first hit, the crystallization conditionswere optimized, and the best crystals were obtained in a condi-tion containing 40% (v/v) PEG 300, 0.1 M sodium cacodylate,pH 6.5, and 0.2 M calcium acetate hydrate. The crystals grew totheir maximum size within 2 days.

The concentrated fiAnGDH (12 mg/ml) sample was incu-bated for 30 min at 25 °C with �-ketoglutarate and NADH toprepare a complex. The final concentrations of �-ketoglutarateand NADH used for preparing this complex were 0.2 and 0.2 M,respectively. The crystallization screens for this complex wereset up, and the best crystals appeared after 2 weeks in a condi-tion containing 0.15 M potassium bromide and 20% (w/v) PEG2000 MME, and grew to their maximum size in 2 weeks.

Data collection and processing

All the diffraction data were collected from the frozen crys-tals by the rotation method. The crystals were briefly trans-ferred to their corresponding cryoprotectant solutions using anylon loop and subsequently flash-frozen in the liquid nitrogenstream at 100 K. A dataset for the apo-AnGDH crystal wascollected using CuK� X-ray radiation source generated by aBruker MICROSTAR diffractometer equipped with MAR345detector at the Advanced Centre for Treatment, Research, andEducation in Cancer, Navi Mumbai, India. The reservoir solu-tion with 20% (v/v) glycerol was used as a cryoprotectantfor freezing the apo-AnGDH crystal. Diffraction data fromfiAnGDH crystals were also collected using CuK� radiationgenerated by a Rigaku Micromax 007HF generator equippedwith R-Axis IV�� detector at the Protein CrystallographyFacility, IIT Bombay, India. The crystals of AnGDH–AKG–NADPH, AnGDH–IPT–NADPH, and AnGDH–AIG–NADP�

complexes were first briefly transferred to the cryoprotectantsolutions prepared from their respective mother liquors con-taining 30% (v/v) glycerol, and then subsequently flash-frozenin liquid nitrogen. The frozen crystals were then transferred to

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the liquid nitrogen stream at 100 K for data collection. Thediffraction data sets from these complexes were collected at theBM14 beamline of European Synchrotron Radiation Facility(ESRF), Grenoble, France, using a MarCCD detector. Indexing,integration, and scaling of all the data sets were performed byXDS (58). The intensities were converted to structure factorswith the program modules F2MTZ and CAD of CCP4 (59). Thedata collection statistics are presented in Table 1.

Structure determination, model building, and refinement

The structure of the apo-AnGDH was determined by molec-ular replacement. The A subunit of E. coli glutamate dehydro-genase (EcGDH) crystal structure (PDB code 3SBO), which hasan amino acid sequence identity of 55% with AnGDH, was usedas the search model. Calculation of Matthews coefficient (2.7Å3 Da�1) (60) indicated the presence of six apo-AnGDH mol-ecules in the asymmetric unit. The correct orientations of sixsubunits were identified by PHASER (61) using the searchmodel. The hexameric unit of the model was refined for 10cycles using REFMAC5 (62). The resulting electron densitymap was used for automated model building using Buccaneersoftware (63), which could correctly assign almost 60% residuesof the hexameric AnGDH structure. The partially built modelwas used for subsequent manual model building by visualinspection in COOT (64) and refinement using REFMAC5. Thesolvent molecules and ions were progressively added at peaks ofelectron density higher than 3� in �-A weighted Fo � Fc elec-tron density maps while monitoring the decrease of Rfree andimprovement of the overall stereochemistry. In the structure,subunits A–C have one and subunits D–F have two N-terminalresidues missing, as they could not be built due to lack of fea-tures in the electron density.

The structures of AnGDH–AKG–NADPH complex andfiAnGDH were solved by molecular replacement using thecoordinates of apoenzyme A subunit. The initial phases ofthe structures of other AnGDH complexes were obtained bythe rigid body refinement of the protein part of the AnGDH–AKG–NADPH structure as all the complexed crystal formshad almost identical cell dimensions and belonged to thesame space group. The first few cycles of refinement of onlythe protein molecules were performed by REFMAC5. Subse-quently, the ligands were placed inside the �-A weightedFo � Fc electron density map, and further refinement cycleswere carried out. The waters and other solvent moleculeswere added to the structures, and alternative conformationsof residues were built using COOT. Convergence of therefinement process was monitored by the decrease of Rfreeand improvement of the overall stereochemistry. The refine-ment statistics of all the structures presented in this studyare reported in Table 1.

Author contributions—P. P. and P. B. data curation; P. P., N. S. P.,and P. B. formal analysis; P. P. and P. B. validation; P. P., N. S. P., andP. B. visualization; P. P. and P. B. methodology; P. P. and P. B. writ-ing-original draft; P. P., N. S. P., and P. B. writing-review and editing;N. S. P. and P. B. conceptualization; P. B. resources; P. B. software;P. B. supervision; P. B. funding acquisition; P. B. investigation; P. B.project administration.

Acknowledgments—We thank the EMBL staff Dr. Hassan Belrhaliand Dr. Babu A. Manjasetty for providing support on the beamlineand EMBL-DBT for providing access to the BM14 beamline at theESRF. We are thankful to Dr. Adhish S. Walvekar and Nupur Agar-wal for their advice on protein purification, enzyme assays, and prep-aration of the forward-inhibited enzyme. We express our gratitude toUlka U. Sawant and Dr. Ashok K. Varma from Advanced Centre forTreatment, Research, and Education in Cancer (Navi Mumbai,India) for providing us with access to the X-ray diffractometer.The “Protein Crystallography Facility” at IIT Bombay is alsoacknowledged.

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Structural insights into the catalytic properties of GDH

6258 J. Biol. Chem. (2018) 293(17) 6241–6258

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Prem Prakash, Narayan S. Punekar and Prasenjit Bhaumikglutamate dehydrogenase

-ketoglutarate cooperativity ofαStructural basis for the catalytic mechanism and

doi: 10.1074/jbc.RA117.000149 originally published online March 14, 20182018, 293:6241-6258.J. Biol. Chem. 

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