Crystallographic study of coenzyme, coenzyme analogue and substrate binding in 6-phosphogluconate dehydrogenase: implications for NADP specificity and the enzyme mechanism

Crystallographic study of coenzyme, coenzyme analogue and substrate binding in 6-phosphogluconate dehydrogenase: implications for NADP specificity and the enzyme mechanism Margaret J Adams*, Grant H Ellis, Sheila Gover, Claire E Naylor and Christopher Phillips University of Oxford, Laboratory of Molecular Biophysics, South Parks Road, Oxford OX1 3QU, UK

Background: The nicotinamide adenine dinucleotide phosphate (NADP)-dependent oxidative decarboxylase, 6-phosphogluconate dehydrogenase, is a major source of reduced coenzyme for synthesis. Enzymes later in the pentose phosphate pathway convert the reaction product, ribulose 5-phosphate, to ribose 5-phosphate. Crystallographic study of complexes with coenzyme and substrate explain the NADP dependence which determines the enzyme's metabolic role and support the proposed general base-general acid mechanism. Results: The refined structures of binary coenzyme/ analogue complexes show that Arg33 is ordered by binding the 2'-phosphate, and provides one face of the adenine site. The nicotinamide, while less tightly bound, is more extended when reduced than when oxidized. All substrate binding residues are conserved; the 3-hydroxyl

of 6-phosphogluconate is hydrogen bonded to Nr of Lys183 and the 3-hydrogen points towards the oxidized nicotinamide. The 6-phosphate replaces a tightly bound sulphate in the apo-enzyme. Conclusions: NADP specificity is achieved primarily by Arg33 which binds the 2'-phosphate but, in its absence, obscures the adenine pocket. The bound oxidized nicotinamide is syn; hydride transfer from bound substrate to the nicotinamide si- face is achieved with a small movement of the nicotinamide nucleotide. Lys183 may act as general base. A water bound to Glyl30 in the coenzyme domain is the most likely acid required in decarboxylation. The dihydronicotinamide ring of NADPH competes for ligands with the 1-carboxyl of 6-phosphogluconate.

Structure 15 July 1994, 2:651-668 Key words: enzyme mechanism, NADP/NADPH binding, 6-phosphogluconate dehydrogenase, substrate binding

Introduction The pentose phosphate pathway enzyme 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) oxidatively decarboxylates 6-phosphogluconate (6PG) to give ribulose 5-phosphate (Ru5P). The enzyme is dimeric and NADP-dependent for almost all species [1]. Seven complete primary sequences have been determined (for sheep [2]; Trypanosoma brucei [3]; Escherichia coli [4]; Salmonella typhimurium [5]; Synechococcus [6]; Bacillus subtilis [7]; Drosophila melanogaster [8]) and two further partial sequences (pig [9,10]; mouse: S Hoffmann, personal communication). The subunit contains between 468 (E. coli) and 482 residues (sheep), 88 of which are conserved in all sequences reported so far. The kinetic mechanism has been studied most intensely for the sheep liver enzyme [11] and for that from Candida utilis [12]. The oxidative decarboxylation reaction has been described as asymmetric sequential for the sheep enzyme with ordered product release: carbon dioxide first and NADPH last. The preferred binding order for NADP+ and 6PG depends upon the buffer system used; for the sheep enzyme in

phosphate buffer, where there is evidence that NADP + may bind before 6PG, the dissociation constant (Kd) for NADP+ is 26~ M [13] and the Michaelis constant (Km ) for 6PG is 313 M. In triethanolamine (TEA) at pH 7.0, the dominant path is through a complex of enzyme with 6PG, and the Km for 6PG was calculated as 23 [M. Product release is ordered in either buffer with carbon dioxide leaving first, followed by Ru5P. 6PGDH oxidizes the 3-hydroxyl of 6PG and decarboxylates the resulting 3-keto,6-phosphogluconate. Multiple isotope effects have been interpreted in terms of a sequential mechanism [14]; however, a recent investigation under a wider range of conditions suggests that an asynchronous concerted oxidative decarboxylation after formation of a C-3 alkoxide cannot be ruled out (P Cook, personal communication). An acid-base mechanism has been proposed; involvement of a Schiff base has been excluded [15] and no metal ions are present, a feature which distinguishes the reaction from those of isocitrate dehydrogenase (IDH) and malic enzyme. Experiments with the sheep enzyme failed to demonstrate activity with NAD+ and indicated that it did not bind (M Silverberg, personal communication); NAD+

*Corresponding author.

( Current Biology Ltd ISSN 0969-2126

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has been shown to act as a poor coenzyme in C utilis 6PGDH with a Km 1000 times that for NADP + [16]. The reduced coenzyme binds more tightly than the oxidized species; for the sheep enzyme in phosphate, Kd at pH 7.0 is 5.7 gM [17]. The affinity of both oxidized and reduced coenzymes is enhanced 10-fold in TEA or in acetate [11]. While most nucleoside phosphates inhibit competitively with both coenzyme and substrate, 2'AMP is competitive with NADP + and non-competitive with 6PG [18]. There is evidence that in some circumstances NADPH has a role in decarboxylation of the oxidized substrate [19]. Inorganic phosphate has been shown to stabilize 6PGDH [20] and to inhibit competitively with both coenzyme and substrate with inhibition constants (K i ) in the 4-8 mM range. Oxaloacetate, citrate, sulphate (Ki 8mM) and pyrophosphate also inhibit [18]. Of the wide range of metabolic intermediates and analogues investigated, 6-sulphogluconate, 6-phosphoallonate, 5phosphoribonate, and 5-phosphoarabonate inhibit the C utilis enzyme and are competitive with substrate while 6-phosphomannonate is a poor substrate [21]. Sheep liver 6PGDH is inhibited by fructose 1,6-bisphosphate (K i 0.07mM), by the 1- and 6-monophosphates and by glucose 6-phosphate; all are competitive to substrate only [18], with Ki values mostly in the millimolar range. The analogue 2-deoxy 6-phosphogluconate [19] is a substrate which gives rise to an intermediate keto-acid which is decarboxylated slowly. The three-dimensional structure of the sheep liver enzyme has been refined at 2.5A resolution [22] and subsequently with data extending to 2.0A (C Phillips, S Gover and MJ Adams, unpublished data). The enzyme crystallizes in the space group C222 1 ; the dimer has crystallographic two-fold symmetry. The monomer is illustrated in Fig. 1 for reference. Each subunit has three domains: the amino-terminal 13-a-3 domain (residues 1-176) has a typical dinucleotide-binding fold [23] followed by a short helix and an additional P-ac-03 unit antiparallel to that fold; the second domain (residues 177-434) is the largest, and is helical; the carboxy-terminal tail (residues 435-482) burrows through the second subunit. Beyond Gly473, the tail is disordered. A tightly-bound sulphate ion was identified in the apoenzyme crystals, which were grown from ammonium sulphate at pH6.5. This sulphate is important in making inter-subunit contacts and is bound by strictly conserved residues in the helical domain of one subunit and in the tail of the two-fold-related subunit. Here we show that crystals grown in the presence of 6PG give a binary complex isomorphous to the apo-enzyme and confirm the suggestion that the 6-phosphate of 6PG displaces the sulphate ion. Two further, less tightly bound, sulphate ions have been recognized in the apoenzyme 2.OA electron density map; one is at a crystal contact. The remaining sulphate, in the same pocket as the first and approximately 6 A from it, is also displaced by 6PG; it makes three contacts to conserved residues

in the same monomer, one from the coenzyme domain and two from the helical domain, and four contacts to bound water molecules. The binding site for coenzyme has been reported at low resolution [24]; it lies at the carboxy-terminal end of the parallel -sheet. This paper describes crystallographic studies to define the binding of oxidized and reduced coenzyme and of reduced substrate at high resolution. Coenzyme or coenzyme analogue has been soaked into crystals and binding has been seen in difference electron density maps. In these experiments, we have used NADPH, the active NADP+ analogue nicotinamide-8-bromo-adenine dinucleotide phosphate (Nbr8 ADP + ) [24] and 2'AMP, since it is the

only metabolite known to be competitive with coenzyme alone. These molecules bind with very similar conformations for the adenine, adenine ribose and 2'phosphate. The oxidized and reduced nicotinamide

rings bind differently: all protein contacts to the oxidized analogue are from the coenzyme-binding domain. The dihydronicotinamide makes contact with residues in the helical domain which bind the 1-carboxyl of 6PG in its binary complex. The structure of the co-crystallized substrate binary complex has shown the general base to be the conserved lysine at position 183. It has further shown that the residues responsible

for the specificity of substrate binding come from both the coenzyme and the helical domains of one subunit, and from the tail of the second.

Results Binary complex structures and refinement

Fig. 2 shows the initial difference maps, contoured at 3o and 60, calculated using terms (Fcomplex - Fnative) exp ioca, where the phase set for the refined native structure, including solvent, was used. There is no evidence in any difference map of changes in protein conformation distant from the coenzyme or substrate binding site. In the 2'AMP complex, there is difference density which corresponds to the complete small molecule; in the two dinucleotide complexes, the adenine, adenine ribose, 2'-phosphate and bis-phosphate are seen. Density corresponding to the side chain of Arg33 is also visible in all coenzyme difference maps; this side chain is disordered in the native protein. There is no difference density above 3 for the nicotinamide ribose or the nicotinamide in any dinucleotide difference map. The main features of the 6PG difference map are a peak in the internal solvent cavity which is smaller than a complete 6PG molecule and a hole corresponding to the position of sulphate 507 in the apo-enzyme. The peak approaches the position occupied by sulphate 505 in the apo-enzyme; there is no difference density at this position. One additional small peak was visible in this map which indicated a change in orientation of the amide of AsnlO02.

6PGDH coenzyme specificity and mechanism Adams et al.

Fig. 1. Monomer of 6PGDH (drawn using MOLSCRIPT [52]) with domains and secondary structural elements labelled; [-strands A-H (blue), ot-helices a-s (red).

The two NADPH complexes are essentially identical if allowance is made for the difference in resolution limit: an electron density map with terms FNADPH(2.5A)-FNADPH(3.OA) and apo-enzyme phases

was flat (the largest peak or hole was < 1.5 % the height of the 2'-phosphate peak in the 3.0A NADPH-apo-enzyme difference map). There was no density corresponding to substrate and it was concluded that the reduced coenzyme had displaced substrate from its binding site. Density corresponding to the nicotinamide ribose and to both the oxidized and reduced nicotinamide rings appeared in their respective omit/2Fo-Fc maps during the course of the simulated annealing refinement. Similarly, the conformation of the 6PG molecule became apparent. In all these maps, the dinucleotide or substrate and solvent molecules within the binding cavity were omitted from the phasing; for the NADPH and 6PG complexes, they were also omitted from refinement (see Materials and methods). In the oxidized coenzyme analogue, extended solvent was interpreted as the competitive inhibitor pyrophosphate [18], which was used in eluting the enzyme from an affinity column [25]. In Fig. 3, the coenzyme and substrate coordinates are superimposed on the final omit/2Fc-Fc maps for the 2.5A NADPH, Nbr 8ADP + and 6PG complexes. For each complex, the substituent and solvent within the binding site were omitted from the phase calculation. In no coenzyme bound structure was the sulphate ion

(505), which is tightly bound between subunits, displaced. For each dinucleotide, the occupancy which yielded appropriate thermal parameters (see Materials and methods) was lower for the nicotinamide and nicotinamide ribose than for the remainder of the coenzyme. Fractional occupancies were set at 0.5 and 0.8 for Nbr 8 ADP + and at 0.5 and 0.7 for NADPH. (No precautions were taken to prevent NADPH oxidation and the lower occupancy of the dihydronicotinamide ring may reflect partial oxidation to NADP+, which has a higher Kd. The short soak time used should diminish the extent of this problem.) The difference in fractional occupancies for the sugar of 6PG (0.75) and for its 6-phosphate (1.0) are a consequence of incomplete substitution of the tightly-bound sulphate ion (505). More than 10 % of the sulphate can be expected to remain bound. Using the solution values for Kd and K, Kd (6PG) = 2 M, Ki (SO42 -) = 8mM, [6PG] = 30mM, [S0 42 -] = 2.1M, the effective ratio (SO42- bound)/( 6 PGbound) = (2 x 10-6/8 x 10-3) x

(2.1/30 x 10-3) = 0.13. Table 1 gives refinement statistics and Table 2 the parameters of the final models, with the coordinate errors derived from the change of residual with resolution [26]. Coenzyme, coenzyme analogue and substrate conformations

The conformations of the adenine, adenine ribose and 2'-phosphate are very similar in the three structures. Although the dinucleotides in both complexes are ex-

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Fig. 2. Initial difference maps for (a) Nbr8ADP + , (b) NADPH 2.5A data (c) NADPH 3.0A data, (d) 2'AMP, and (e) 6PG. The final refined coordinates of the ligands are superimposed. The refined coordinates of Arg33, which is seen to move in coenzyme complexes, are superimposed in (a), (b), (c) and (d). In (b), the pyrophosphate (PPH506) is drawn. The similarity of the two NADPH difference maps demonstrates that the 2.5 A data set is of a binary complex. In (e), the two sulphate ions which occupy the site in the apo-enzyme and are displaced by 6PG are shown. In this figure, both positive (white) and negative (red) contours are drawn. All maps used apoenzyme phase sets including active site solvent; they are contoured at 3a and 6a. Standard deviations () were calculated using the standard errors of the input data [53].

tended, the nicotinamide ribose and nicotinamide of the oxidized and reduced dinucleotides have different conformations. The greater extension of the reduced coenzyme can be seen in Fig. 4. The adenine ring is anti for all three complexes. While the plane of the nicotinamide ring is close to that of the nicotinamide ribose in both dinucleotide complexes, the conformation is essentially syn in the Nbr8ADP + complex. The adenine ribose is C2'-endo (C3'-exo) in all three complexes; the nicotinamide ribose is C3'-endo in the NbrsADP + complex and close to C2'-endo in the NADPH complex. The conformation of the bound 6PG is shown in Fig. 5, where it is compared with the small molecule structure (tri-sodium salt). The root mean square (rms) difference in the superimposed coordinates is 1.49k The most striking difference between the conformations is the orientation of the 1-carboxyl with respect to the carbon backbone. Binding sites

The mean differences of positional parameters between the apo-enzyme and the protein in each com-

plex are given in Table 3, for all residues and for those within 10A of the substituent. There is little protein movement required to accommodate the coenzyme other than the ordering of the side chain of Arg33 and a small movement of some of the residues in two loops: 73-78 (3D-ad) and 130-133 (PF-a-f). The pattern of main-chain temperature factors is similar to that of the apo-enzyme. Many of the well-bound waters are common to apo-enzyme and complexes; details of the number and proportion in common are given in Table 3. The coenzymes replace ordered waters: 11 waters and a sulphate are displaced by the nicotinamide nucleotide of NADPH, 2 waters by the adenine nucleotide. Twelve of these waters, and two extra ones, are displaced by Nbr8 ADP +; the sulphate and one remaining water are replaced by the pyrophosphate. On binding coenzyme, the solvent accessibilities of several residues in the binding site are decreased. These residues are: Alal 1, Metl3, Arg33, Lys37, Leu73, Val74, Lys75 and Ala79. The hydrophobic residues amongst these are more exposed than the average of their types in the apo-enzyme and contribute towards an exposed hydrophobic patch.

6PGDH coenzyme specificity and mechanism Adams et al.

Table 1. Refinement statistics. Complex

Resolution Final limits residual

Parameters of solvent maska

Nbr8ADP+

20-2.3 A

a = 53.37 %

20.4 %

Other comments

B = 90A2 -3

NADPH (2.5 A)

20-2.5 A

2'AMP

20-3.17 A

6PG

20-2.5 A

17.0%

16.9 %

r = 0.390eA a = 53.51% B = 90 A2 r = -0.385 e A-

3

a = 53.71% B = 95 A2 - 3 r = 0.370e A 17.0 % a = 53.83 % 2 B = 95 A 3 r = 0.385eA-

24 residues, sulphate 505 and 2 waters constrained and omitted/61 residues and 21 waters restrained in intermediate cycles No temperature factor refinement 20 residues and 1 water constrained and omitted/50 residues and 14 waters restrained in intermediate cycles

aa = % solvent; B = temperature factor; r=bulk solvent density based on fractional occupancy of water. These parameters are defined by Brunger [43].

In the 6PG complex, the substrate binds to the protein with only a small movement of protein residues. The most important change is the removal of the two sulphates and four waters in the active site pocket. Six waters remain within 5A of the substrate in the active site pocket; four of these are also present in the apoenzyme. Contacts to protein and probable hydrogen bonds are illustrated in Fig. 6 for each nucleotide complex and for the 6PG complex. Potential hydrogen bonds to the nucleotides are listed in Table 4. The pattern of contacts and hydrogen bonds for adenine, adenine ribose and 2'-phosphate is similar in all three complexes. Residues in contact with the different parts of the substrate are indicated in Fig. 7. Potential hydrogen bonds are listed in Table 5 where they are compared with the contacts to the two inorganic sulphate ions in the apo-enzyme.

Fig. 3. Final omit/2Fo-Fc maps for (a)Nbr 8ADP + , (b) NADPH 2.5 A data and (c) 6PG data. The coenzyme or substrate and solvent molecules in the binding sites were excluded from the calculation of Fc. The coenzyme is in the same orientation as in Fig. 2. Final refined coordinates are superimposed with coenzyme (and pyrophosphate) and substrate in atom colours and protein in red. These maps are contoured at 1.5cr and 3.

Adenine nucleotide contacts The adenine is bound in a pocket, one side of which is formed by side chains of hydrophobic residues in the loop between D and ad (Val74 and Ala79) and in d (Phe83). The depth of the binding pocket is limited by Phe83, which defines its end. The side chain of Arg33, which is only ordered in these binary complexes, forms the other side of the pocket, shielding the adenine from solvent. The plane of the guanidinium group is approximately parallel to that of the adenine ring. There is a small additional movement of Phe83 in the Nbr8 ADP + complex to accommodate the larger brominated adenine. The bromine extends into solvent

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Table 2. Model parameters. Complex Standard deviation from ideal: Bond lengths Bond angles Dihedral angles Improper angles Mean B factor: Main chain Side chain Waters included: Full: number, mean B Half: number, mean B Number (%) residues with [,, in Ramachandran plot: Most favoured regions Disallowed regions [481 Mean coordinate error (from Luzzati plot [261)

NbrADP+

NADPH

2'AMP

6PG

0.011 A ° 2.66 ° 23.0 1.87'

0.011 A ° 2.53 ° 23.0 ° 1.35

0.043 A 2.75' ° 22.9 ° 1.73

0.011 A ° 2.37 ° 21.9 1.30

32.6 A 33.9 A2

35.1 A2 37.6 A2

(27.5 A2) a (31.3 A2)

30.1A2 2 32.2 A

320 43.5A2 100 36.3A2

148 45.9A2 271 48.7A2

421 62.9A2b

318 48.3A2 80 38.5A2

351 (91.6 %) 2 (0.5 /%)

358 (93.5 %) 2 (0.5 %)

348 (90.8 O/o) 1 (0.3 %)

354 (92.9 %) 2 (0.5 %/o)

0.30-0.35 A

0.25-0.30 A

0.25-0.30 A

0.20-0.30 A

2

aTemperature factors were not refined in this structure. bWaters were not refined in- this structure but native waters were included at an intermediate stage of the refinement before partial occupancies were assigned.

Fig. 4. Stereo pair showing the confor-

mation of NADPH (red) and Nbr8 ADP + together with pyrophosphate (blue) when bound to 6PGDH. Atom types are indicated by size: C < N < O < P < Br.

Fig. 5. Stereo pair showing the conformation of 6PG in the structure of the tri-sodium salt (red) and as bound to 6PGDH (blue). Atom types are indicated by size: C
and is not responsible for any other perturbation of the enzyme structure. There are potential hydrogen bond interactions to the 2'-phosphate in all complexes from the side chains of three residues in the loop between PB3and cab: Asn32, Arg33 and Thr34. The interactions involve at least three of the phosphate oxygens with often more than one hydrogen bond being possible to each. The hydrogen

bonds are made by the y-hydroxyl of Thr34 and both the oxygen and nitrogen of the amide of Asn32. In both dinucleotide complexes, the adenine ribose and bis-phosphate make van der Waals contact with

three residues of the tight 3A-aa turn (Gly9, LeulO and Alall) and with Val74 and Lys75 of the 3D-ctd turn. The 3'-hydroxyl of the adenine ribose is within hydrogen-bonding distance of the main chain nitrogen of LeulO and the main chain nitrogen of Lys75 can

6PGDH coenzyme specificity and mechanism Adams et al.

Table 3. Structural comparison of complexes with apo-enzyme. Coenzyme

Waters in common with apo-enzymea

Mean positional difference All residues Residues within 10A mc

sc

mc

sc

Nbr8 ADP +

0.249 A 368 atoms

0.413 A 332 atoms

0.236 A

0.566 A

126 (30 %)

NADPH

0.230A 368 atoms

0.535 A 332 atoms

0.188A

0.488A

130 (31%)

6PG

0.219A 272 atoms

0.508A 236 atoms

o.199A

0.492 A

161 (40 %)

Residues within 10A with large positional differencesb mc

sc

74, 75, 76, 77, 78 (130), (131), 132, 259, 260 (74), (75), (131), 132, (133), (260), (262), (450) (75), (132), (447), (448)

33, 75, (260)

33, (73), (74), 75, (260) (73), 75

6 aWaters are considered to be in common if they are less than 1.2 A distant and at least 0 % of the protein contacts within 3.2 A are the same. bLarge differences in main chain (mc) correspond to mean differences > 0.5 A (0.4 A); those in side chain (sc) correspond to mean differences > 2 A (1.5 A).

hydrogen bond with the ring oxygen of the adenine ribose. In all complexes, 061 of Asn32 also interacts with this 3'-hydroxyl group. Nicotinamide nucleotide contacts

The remaining interactions differ between the oxidized and reduced coenzyme complexes. In the complex with the oxidized analogue, Lys75 extends to follow the shape of the nicotinamide ribose and van der Waals interactions are also made with the loop between PE and ae (residues 100-104). AsplO02 in this turn makes van der Waals contact with the ribose and is completely conserved in all seven species. Almost all interactions with the nicotinamide are made by the conserved Metl3; the thioether is above the nicotinamide ring. The only additional direct hydrogen bonds to protein from the oxidized coenzyme complex are made by the nicotinamide amide: the oxygen with the main chain of Metl3 and the -NH2 group with the carboxyl of Glu131 at the beginning of af. The pyrophosphate ion, which replaces sulphate 507 in this complex, interacts with Ns of Lys183 (in h), the amide of AsplO02, the main chain nitrogen groups of the residues of the tight ,F-af turn (Gly129 and Gly130), and with two waters (614 and 886) which are present in the apo-enzyme structure. The charge of the pyridinium ring is counterbalanced by the bound pyrophosphate ion. In the NADPH complex, the nicotinamide ribose, instead of interacting with Lys75, contacts N82 of Asnl02 in the loop between E and ae. The bis-phosphate is hydrogen-bonded through water 589 to the main chain nitrogen of both Alall and Gly14 (residues at the beginning of ca) and to the carbonyl oxygen of Leu73 (in the fID-cd loop), through water 692 to the main chain nitrogen of Metl3 and through a third water (516) to the main chain nitrogen of AsnlO02. All three waters are also present in the apo-enzyme structure. Instead of Metl3, residues from the PF-af turn, Val127, and the totally conserved Serl28, Gly129 and Glyl30 interact with the nicotinamide. The nicotinamide amide

hydrogen bonds Oy of Ser128 at the end of PF, NE2 of His186 and 061 of Asnl87; both these last residues are in the first helix of the helical domain (ah) and are conserved for all known sequences. Additional conserved residues from this helix (Lys183 and Glu190) make van der Waals contact, primarily with the amide function. Protein-substrate contacts

The inter-subunit nature of the substrate site is immediately apparent, as is the very large proportion of totally conserved residues within 4A of 6PG. The substrate makes hydrogen bonds to residues in the F--af loop of the coenzyme domain and to residues in three regions of the helical domain of one subunit: one face of the helix ah, the end of the long cj-ak loop and Arg287 in al. Hydrogen bonds are also made to residues in the tail of the second subunit of the dimer, in cts and in the turn immediately following it. The interactions to the 6-phosphate of 6PG are the same as those to sulphate 505 in the apo-enzyme. There is no direct match between sulphate 507 and the carboxyl of 6PG; the carboxyl interactions match the contacts to water 614 of the apo-enzyme most closely. This water forms a hydrogen bond to sulphate 507, as does water 613 which is also displaced by the substrate. There is a considerable overlap between residues interacting with the carboxylate and those interacting with the reduced nicotinamide. His186 is within 4A of the carboxyl and, if it is protonated, will serve to balance the negative charge. The carboxyl group of the substrate bonds to Oy of Serl28 and to 0O1 of Glul90; the latter had no close neighbour in the apo-enzyme. The second oxygen of the carboxyl group of 190 hydrogen bonds the conserved water 528 in all complexes and in the apoenzyme. This water makes two additional hydrogen bonds: to Arg287 Nq2 in both structures; and to 02 of the 6-phosphate of 6PG or to the equivalent oxygen (03) of sulphate 505. The 2-hydroxyl (08) makes direct hydrogen bonds to two waters; water 1232 in

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Fig. 6. Stereo pairs showing the binding mode of (a) Nbr8ADP+, (b) NADPH, (c) 2'AMP and (d) 6PG. The ligand and protein atoms are shown in red and blue, respectively. Potential hydrogen bonds are indicated by broken lines. Note that the oxidized (a)and reduced (b)coenzymes have different conformations with the nicotinamide moiety of NADPH binding to residues from both domains and seen in (d)to be involved in substrate binding. Lys75 and Met13 play different roles in binding the two coenzymes. Substrate binding is seen in (d) to be dominated by recognition of the 6-phosphate; the charge is balanced by two arginines: Arg287 and Arg446 of the two-fold related subunit. The sugar conformation is determined by a hydrogen bond network linking the substrate carboxy and phosphate groups via Glu190 and an active site water.

6PGDH coenzyme specificity and mechanism Adams et al.

Fig. 7. Residues in contact with the different parts of the substrate, 6PG. Hydrogen bonds are indicated by full lines; other possible hydrogen-bonded or polar interactions by broken lines; # indicates residue from two-fold related subunit.

turn contacts the main chain nitrogen of Gly130 of the OF-af turn; water 1109 has no other ordered interactions. In contrast, the 6PG 3-hydroxyl makes two direct hydrogen bonds to protein: to N of Lys183 and to N62 of Asn187. While the C4 and C5 hydroxyls do not make good hydrogen bonds in the binary complex, His452 of the second subunit approaches them and a small change in conformation would allow at least one hydrogen bond to be made. These hydroxyls are constrained by the tight interactions of the 6-phosphate and the oxygens of C1, C2 and C3.

Discussion Comparison of coenzyme conformations in binary complexes The experiments described above show that 2'AMP, the reduced coenzyme and an active oxidized coenzyme analogue, modified at the adenine, will bind to apo-6PGDH in the crystal without invoking any large conformational change of the enzyme. Both coenzymes bind in an open conformation, but the conformations beyond the bis-phosphate are distinct. The reduced coenzyme in the 6PGDH complex is as extended as is the coenzyme in dihydrofolate reductase (DHFR). The distance between C6 of the adenine ring and C2 of the nicotinamide ring is usually taken as a measure of this extension. For most bound coenzymes, the distance is between 14A and 15A; for NADPH bound to DHFR, it is 17.1A [23]. The distance for Nbr8 ADP + bound to 6PGDH is 15.1A; that for bound NADPH is 17.8k The conformations of 2'AMP and the adenine mononucleotide moieties of the two dinucleotides are closely similar. The anti conformation of the adenine ring is anticipated for the unsubstituted adenine moieties, but for the Nbr8 ADP+ complex it contrasts with the syn conformation preferred in free 8-bromo-adenosine [27]. The anticonformation of the 8-bromo-ade-

nine ring has also been observed when nicotinamide8-bromo-adenine dinucleotide is bound to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [28] and in other enzyme complexes. The 6PGDH coenzyme site, in common with those of other dehydrogenases, is thus specific for the anti conformer. The close superposition of 2'AMP with the comparable fragments of the dinucleotides is consistent with inhibition competitive only with coenzyme. Specificity of protein for NADP; importance of Arg33 Discrimination between NAD and NADP binding requires that the energy of the 2'-phosphate-protein interaction is a large proportion of the total binding energy. This may be achieved by a large net 2'phosphate-protein binding energy, a small net binding energy of the remainder of the coenzyme, or both. The 2'-phosphate dominates in the hydrogen bond interactions made directly to the protein (shown in Table 4), taking part in 7 of 14 direct interactions of NADPH, 6 of 11 interactions of Nbr 8ADP + and 5 of 7 interactions of 2'AMP. Of the three consecutive residues in the turn between B and acb making these hydrogen bonds, Asn32 is conserved in all seven known sequences, Arg33 is replaced by tyrosine in one sequence (that of B. subtilis) and Thr34 is replaced by serine in both the E coli and S. typhimurium sequences. Identical hydrogen-bonding potential is thus retained at residues 32 and 34 and a planar residue with capacity to act as a hydrogen bond donor at 33. The dominance of the 2'-phosphate interaction must make a major contribution to the NADP + specificity of this enzyme. The position of Asn32 in the turn between ,B and ab is the same as that of the aspartate which hydrogen bonds the adenine ribose in NAD-dependent dehydrogenases [29]. In 6PGDH, acb is oriented and positioned so that its amino terminus is approximately 5A from the 2'-phosphate and the helix dipole may contribute to stabilizing the negative charge.

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Table 4. Hydrogen bonds to coenzyme.a

Coenzyme moiety

NbrSADP+

Table 5. Hydrogen bonds to substrate. NADPH

2'AMP

Adenine

03-NH

Leu10

03-NH

Leu10O

ribose

03-N62

Asn32 Lys75S

03-O1 04-NH

Asn32

03-N62

Asn32'

Lys75

04 NH

Lys75

04-NH 2'-phosphate

02(R)-N82 Asn32* 01(P)-NT2 Arg33 02(P)-061 Asn32' 02(P)-Nc Arg33 02(P-Oyl Thr34 03(P)-O1 Asn32* ' 03(P)-N82 Asn32 b

Bis-phosphate

6PG region

6PG complex

Inorganic ion

1-carboxy (09, 010)

O10-0y 5er128

50 4 507

09-OC1 Glu190'

02(R)-Nrl Arg33b

01(P)-NE Arg33 01(P)-N12 Arg33 02(P)N62 Asn32' 02(P-NH Thr34 02(P)-Oyl Thr34 * 03(P-061 Asn32

C2-C6a 02(P-Nll 02(P) Oy1 03(P)-01 03(P)-N82

Nicotinamide

d

O1" O

Wat692

02"-0 05(R)-O

Wat589 Wat516b

03-O I 03 NH

04-N2

Asn102*

03-Oy1 Thr262 02 Nll1 Arg287*

07-NH

Met13*

07-Oy

N70-OE2

Glu131'

07-Nc2

Ser128' His186'

N7-061

Asn187'

Pyrophosphate 01'-061 01'- N2

6-phosphate

S0 4505 Tyr191* Lys260*

02'-Nr 02'-0

Waters

Lys183' Wat614

04'-0 02" 0

Wat886c

02"-Nr 02"0

Lys183' Wat614

04"-NH 04" NH

Gly129* Gly130'

0614-OY

Wat1109 Wat1232

Val127

Ser128' 0614-0£1 Glu190' 0886-NE2 His452# 0886-04 504505

03-0 04-0

Wat886b Wat699

01-0 02-OT0 02 NH 02-0

Wat886

Asn102* Asn187*

Tyr191* Lys260' Wat953

03-NTrl

02-Nfll Arg446*# 02-0 Wat528

Arg28703 Nill Arg446*# 03-0 Wat528

01-Nfll Arg446*#

04-Nfl1

Arg446*#

0-062

Glu190

O-NTl2

Arg287

(0-03 O0NH

S0 4505) Gly129

O NH

Gly130*

(0-01 0 0

504507) Wat614

(0-04 O-Oy 0 0 O-NE2

504507) Ser128* Wat613 His186

Water neighbours Wat528

Asn102* Asn102*

Lys183' Wat613 Wat614

Watl109 Wat1232

*b

Nicotinamide ribose

01 Nr 01-0 01-0 02-N62 02 N82

Lys183' 07 Nc 07-N82 Asn187* C 05- N2 His452'#

Asn32* *b Asn32

c

08-0 08-0

Arg33 Thr34

Apo-enzyme

Water 0-0£2 Glu190* O N13 (0-02 (0-08 O-NH (0-08

Arg287* 6PG) 6PG) Glyl130 6PG)

neighbours Wat528

Wat613

c

Wat614

0516-NH

Asn102*

0589-NH

Alall

Wat699

(0-01 0-0

S0 4 507) Va1127

0589-NH 0589-0

Gly14* Leu73

Wat886

(0-04 0-No2

504507) His452'

0589-0 0692 NH

Gly9'(? Met13

(0-01 (0-03 O-NH

504 505)

Wat953

O-Oyl 0-02

Thr262S0 4505

aContacts with donor-acceptor distances 3.3A or less are defined as potential hydrogen bonds. Longer distances (b = 3.4 A; c = 3.5 A) are indicated in the table. Less satisfactory angles are indicated by (?). Totally conserved residues are indicated by (), two-fold related subunit by (#), water by Wat.

Although Arg33 is conserved in only six of the seven known sequences, it is clear that it has a crucial role in determining specificity. This residue orders on binding coenzyme and, as well as binding the 2'-phosphate and providing a charge balance, it is responsible for all the contacts to one face of the adenine ring; the guanidinium group and the adenine ring are stacked and approximately coplanar. The depth of the adenine cleft in sheep 6PGDH is limited by the phenylalanine side chain of residue 83 in ctb. This restricts the possibilities for the adenine site and contributes towards placing the adenine nearer the surface of the enzyme than it is in the NAD dehydrogenases. Fig. 8 illustrates the difference the ordering of Arg33 makes to the adenine pocket, showing that the most favoured confor-

)

50 4 50 7 b Thr262'

Totally conserved residues are indicated by (*), two-fold related subunit by (#) and water by (Wat). aC2-C6 of 6PG: -C2H(08H)-C3H(07H)-C4H(06H)-C5H(05H)C6H 2-. bProbable hydrogen bond 3.5 A long. CProbable hydrogen bond, less satisfactory angle. do1, 02, 03, 04 of sulphate 505 correspond to 04, 03, 02, 01 of 6PG 6-phosphate.

mation of the arginine in the apo-enzyme makes van der Waals contacts with some of the residues which form the hydrophobic contacts to adenine. A similar adenine-arginine interaction has been observed in the NADP-dependent glutathione reductase (GR) [30] and arginine is involved in stabilizing the adenine and 2'phosphate of NADP in avian DHFR [31] and in catalase [32]. This interaction may be identified as one of the common solutions to the problem of enhancing specificity for NADP relative to NAD. It is likely that the driving force for ordering the arginine in 6PGDH is the phosphate interaction and that, in the absence of the 2'-phosphate, there is no adenine cleft.

6PGDH coenzyme specificity and mechanism Adams et al.

Fig. 8. Stereo pair showing Arg33 in its conformation in the apo-enzyme (blue) and in binary complexes (red), and demonstrating its role in forming the adenine pocket.

In the 6PGDH apo-enzyme, the amide of Asn32 forms a bridge between the 3A-ca turn and the turn from B1to ctb; it accepts a hydrogen from the main chain nitrogen of LeulO in a hydrogen bond and donates a hydrogen bond to the main chain carbonyl of Thr34. On binding dinucleotide, the asparagine side chain twists slightly, and it is possible for the amide oxygen to accept a hydrogen from the 3'-hydroxyl of the adenine ribose and the amide -NH 2 to donate a hydrogen to a 2'-phosphate oxygen. The main chain nitrogen of LeulO is also close enough to the 3'-hydroxyl to donate a hydrogen in a hydrogen bond interaction. The hydrogen bonds seen for the asparagine side chain in the apo-enzyme are also retained in the binary complexes. The small degree of hydrogen bond interaction of the bis-phosphate, direct or indirect, is surprising. In both dinucleotide complexes, the negative charge is stabilized by the dipole of the first helix of the dinucleotidebinding fold (a). The closest oxygen (in the second phosphate) is 4A from the first turn of the helix in the reduced complex and 5.5A away in the oxidized complex. The bis-phosphate of NADPH is hydrogen bonded through two waters to the main chain nitrogens of the tight A-aa turn and through a third water to the main chain nitrogen in the 3D-cad turn. The fA-caa turn is a part of the NAD(P) fingerprint [29,33]; the sequence for sheep 6PGDH is compared with the consensus sequences in Fig. 9. The position P3 in the fingerprint is normally glycine; any side chain of this residue would project into the coenzyme-binding site and the C of Alall can be seen in Fig. 6 making contact with the bis-phosphate. This interaction, which renders the amino-terminal turn of ctb less accessible to coenzyme, is undoubtedly responsible for the small number of hydrogen bond interactions of the bis-phosphate; it would lower the affinity of the enzyme for NAD, thus further enhancing the NADP specificity of sheep 6PGDH. Comparison with other NAD(P) enzymes Two possible modes of recognition of coenzyme by the dinucleotide-binding fold have been described by Baker et al. [34]. They focus on the conserved glycine-rich turn of the fingerprint and on the aspartate [conserved in the sequences of lactate dehydrogenase

Position in fingerprint P1 Consensus sequence NAD Gly Consensus sequence NADP Gly Sheep sequence 6PGDH Gly Residue number 9 Secondary structure PA -

P2 P3 P4 X Gly X X Gly X Leu Ala Val 10 11 12 turn ,---

P5 P6 X Gly X Ala Met Gly 13 14 aa -

Fig. 9. NAD(P) fingerprint. Residues in the sheep enzyme that correspond to the fingerprint are shown in bold.

(LDH), alcohol dehydrogenase (ADH) and GAPDH] at the end of the strand B. Direct recognition, seen in glutamate dehydrogenase (GDH), involves a hydrogen bond from the main chain nitrogen of P2 to the 3'hydroxyl of the adenine ribose; for indirect recognition, a hydrogen bond from the main chain nitrogen of P2 is made to the carboxyl of the conserved aspartate and this carboxyl hydrogen bonds both 02' and 03'. In 6PGDH, Asn32 occupies the position in the fold of the conserved aspartate. The hydrogen bond interactions have.features in common with both direct and indirect recognition and the main chain torsion angles are a compromise between the two patterns (Fig. 10). This suggests a range of interactions specifying recognition between the two extremes; it also suggests that the sixth residue in the fingerprint (glycine in LDH, ADH, GAPDH and 6PGDH; alanine in GDH and in one domain of GR) is not the only determinant for the recognition pattern. It is also clear from 6PGDH that an alanine in position P6 of the fingerprint is not a universal discriminator for NADP rather than NAD as proposed by Scrutton et al. [35]. Nicotinamide binding sites; implication for catalysis The conformations of the two dinucleotides bound to 6PGDH involve different interactions with the protein. The larger number of hydrogen bond contacts made by the reduced coenzyme is consistent with its tighter binding to the enzyme in solution. The oxidized coenzyme makes contacts only with residues in the coenzyme domain, but the nicotinamide of NADPH interacts with conserved residues in cah of the helical domain. In the substrate complex, these residues interact with substrate; in the oxidized coenzyme complex, their interaction is with the pyrophosphate ion which has replaced sulphate 507.

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Structure 1994, Vol 2 No 7

180-

I

-

15 12 9-1

Modelled ternary complex and proposal for oxidation 6PGDH is a pro-S (B) dehydrogenase, as is GAPDH [37]. The conformation of the oxidized coenzyme in 6PGDH resembles that of the NAD + in GAPDH [38] quite closely, with an rms difference for all atoms of 1.66A (Fig. 11). The (pro-)chirality of the nicoti-

6 3

-3 -6 P+

-90 -12

namides of the bound dinucleotides in the two oxidation states is different. In solution, pro-S enzymes

O

-15 -18 . -180

'-120

A -60 XA

0

.

60

AA

120

reason is now clear: NADPH displaces the reduced 6PG from its binding site by competing for some of its protein ligands. There is already evidence that the reduced coenzyme has a role in decarboxylation or product release [19] and these results give additional weight to this evidence.

·-

180

PHI

Fig. 10. Main chain torsion angles for the fingerprint region of several NAD(P) and FAD enzymes (after [34]). Data were taken from PDB files except for that of glutamate dehydrogenase. A, 1PGD (NADP) 6PGDH apo-enzyme [221; B, (NADP) 6PGDH-Nbr 8 ADP+ complex (this paper); C, (NADP) 6PGDH-NADPH complex (this paper); D, 1LDM (NAD) lactate dehydrogenase [501; E, 1GD1 (NAD) glyceraldehyde 3-phosphate dehydrogenase [38]; F,1GRA (NADP) glutathione reductase [30]; G, 1GRB (NADP) glutathione reductase [30]; H, (NAD) glutamate dehydrogenase [34]; K, 1PHH (FAD) p-hydroxybenzoate hydroxylase [511; M, 1GRB (FAD) glutathione reductase [301.

The difference in conformation is first apparent at the nicotinamide ribose. The hydrogen bond interaction made with the reduced coenzyme is not made with the oxidized coenzyme and it may be speculated that this difference is a consequence of the charge difference at the nicotinamide ring. The residues proposed as forming hydrogen bonds to the nicotinamide amide are from different domains in the two complexes. Two of the interactions with the reduced coenzyme are with residues which bind the substrate in the 6PG binary complex (Ser128 and Asn187). All residues involved in the nicotinamide interaction in both oxidized and reduced coenzyme complexes are totally conserved. The apo-enzyme has been used as a test case for development of a benzamide probe in the program GRID [36]; the position of the reduced nicotinamide is close to the energy minimum found, which is rather sharp and shows the amide of the probe interacting with His186 and Glu190 (P Goodford, personal communication). His186 is seen to hydrogen bond the nicotinamide amide in the reduced coenzyme complex; Glu190 is one turn below Asn187 in ah and is less than 4A from the nicotinamide. The reduced coenzyme shows mixed inhibition with respect to the reduced substrate [11]. In the soaking experiment which led to the 2.5A NADPH data set, we aimed to prepare an abortive ternary complex: enzyme-reduced coenzyme-reduced substrate but a coenzyme binary complex resulted from this soak. The

have been shown to have the syn conformation. In the 6PGDH-NADPH complex, the coenzyme is positioned where it is argued it does not have a redox function and the ring is anti. In the binary complex with the oxidized analogue, the ring is however closer to the syn than the anti conformation; the si- face of the ring is oriented towards the pyrophosphate ion and the substrate binding cleft. The positive charge is stabilized by the pyrophosphate ion. The nicotinamide ring may be further stabilized in this position by interaction of its it-electron density with the polarizable sulphur of the conserved Met13. Superimposition of the structure of bound Nbr 8ADP +

onto the substrate binary complex shows the bound substrate approaching the si- face of the nicotinamide. Direct hydrogen transfer to NADP + could be achieved, without protein movement, by adjustments of torsion angles of the nicotinamide nucleotide portion of the coenzyme. The Nbr8 ADP + conformation in its binary complex is compared with a possible active conformation, which results from a rotation of 57 ° about the

C5'-C4' bond of the nicotinamide ribose and smaller rotations about the phosphate bonds, in Fig. 12. The modelled active complex, in which the distance from the 6PG hydrogen to C4 of the nicotinamide is 3 A is shown in Fig. 13. Fig. 14 shows the steps of the general base-acid mechanism. The 3-hydroxyl of 6PG should approach the general base when it binds to the enzyme. In the binary complex, the 3-hydroxyl makes hydrogen bonds to N5 of Lys183 and to N62 of Asnl87: NS of the lysine moves by less than 1 A from its position in the apo-enzyme and the asparagine 061 by 0.5A; N62 of residue 187 does not move significantly. (The distinction between N62 and 061 of 187 is apparent in the apo-enzyme where the nitrogen acts as hydrogen bond donor to an oxygen of sulphate 507.) Thus, N62 of 187 acts as donor in the hydrogen bond to the 3-OH (07) of 6PG; the 3-OH must then donate in the hydrogen bond to Lys183 N; which can then be seen to be deprotonated. In the apo-enzyme and in the Nbr8ADP + complex, Lys183 is protonated and interacts with sulphate 507 or with the pyrophosphate which replaces it. The protonation state of Lys183 therefore changes when sub-

6PGDH coenzyme specificity and mechanism Adams et al.

Fig. 11. Stereo pair showing the conformation of Nbr8ADP + as bound to 6PGDH (red) compared with that of NAD+ bound to GAPDH (blue). Atom types are indicated by size: C
Fig. 12. Stereo pair showing the conformation of Nbr8ADP+ as bound to 6PGDH in the binary complex (red) and as modelled in the optimum position for hydride transfer (blue). Atom types are indicated by size: C < N < O < P < Br.

Fig. 13. Modelled active complex showing 6PG and the nicotinamide ring of Nbr 8ADP + (red) and active site residues (blue). Lys183 is labelled. Also shown (clockwise from 183) are: Glu190, water 528, His452 (from the two-fold related subunit), Met13, Glu131, Gly130, Gly129 and His186. C3 of 6PG and C4 of the nicotinamide ring, the atoms involved in hydride transfer, are labelled.

strate is bound and this residue is the best candidate for the base in the oxidative stage of the mechanism. The suggestion that a C-3 alkoxide intermediate may be formed prior to oxidation is consistent with involvement of lysine as the base. Decarboxylation is facilitated by an acid which will polarize the 3-keto group to yield an ene-diol (or diolate) intermediate. The same lysine residue is able to act as acid in this stage of the reaction.

Decarboxylation The enol-keto tautomerization is the final stage of the enzyme reaction and it requires a further acidic group not far distant from C2 of 6PG. Before this stage of the reaction, or concomitantly with it if the reaction is concerted, the carbon dioxide product will leave the binding site. The hydrogen bonds to OF1 of Glu190 and to Oy of Ser128 will be broken. Although there is

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Structure 1994, Vol 2 No 7

,H:B

,H:B

A~ .

B.-O : B:H'

H-

H

2

:-OH 2

H2 OP0 3 -

C02 --

IH 2 OH

1

c02

:B

B H---

NADPH

NADPH N

-OH -OH H2 OPO3

2'

no evidence for a kinetically important conformational change, the possibility of a local structural rearrangement, particularly one involving coenzyme, cannot be ruled out. The structure of a product complex or of a complex with a ribulose 5-phosphate analogue is required before the second acidic group can be identified definitively, but probable candidates are apparent in the 6PG binary complex. Water 1232, one of the two waters which form hydrogen bonds to the 2-OH of 6PG, is a possible general acid. This water contacts, and is probably hydrogenbonded to, the hydrogen of the main chain amide group of the conserved Glyl30; hydrogen bond donation from the protein would contribute to the water's enhanced acidity. An alternative hypothesis involves the conserved glutamic acid at position 190 as a general acid. The carboxyl group of this residue is hydrogen bonded to the carboxyl of 6PG; one of these carboxyls must be protonated. Diffusion away of the product CO 2 removes the requirement for the glutamate to be protonated and it could act as acid in the final tautomerization. The substrate binary complex structure, however, argues against direct involvement of the glutamate since the carboxyl oxygens are more than 5A from C2 of the substrate and any closer approach would be at the expense of the interaction with water 528. The more likely role for a glutamate with a raised pKa is that of enhancing binding specificity for the substrate carboxyl. Role of some conserved residues in binding and catalysis The phosphate group of 6PG makes close contacts with the two conserved arginines in the binding site: residue 287 and residue 446 of the second subunit of the dimer. These arginines balance the phosphate charge and provide specificity. The network of hydrogen bonds from Arg287 through water 528 to Glu190 and the 1-carboxyl of 6PG is important in defining the bound 6PG conformation.

Fig. 14. Steps of the general base-acid mechanism for 6PG oxidative decarboxylation by 6PGDH (after Berdis and Cook [21]).

There are two conserved histidines in the binding site: residue 186 and residue 452 of the two-fold-related subunit. Neither histidine is in a suitable position to act as base (or as acid) in the mechanism. His186 interacts with Ser128 which stabilizes the 1-carboxylate of 6PG; NE2 of the histidine is 3.6 A from one of the carboxyl oxygens. If His186 is protonated it balances the carboxylate charge; if neutral, it would have only a secondary role in substrate binding. The distance between either ring nitrogen and the 3-hydroxyl or the 2-carbon of the substrate is more than 5.5 A and His186 could only act as base or acid if there were a significant conformational change. His452 from the two-fold-related subunit makes its closest approach to the C4 and C5 hydroxyls, contributing towards specificity of the site; it is more than 6A from the 3-hydroxyl or 2-carbon and some 5.5A from the modelled nicotinamide position. The several chemical modifications which appeared to indicate that histidine was essential are likely to have obstructed the substrate-binding site. The side chains of AsnlO2, Ser128, Tyrl91 and Thr261 all contribute to binding specificity by direct interactions with substrate or by forming hydrogen bonds to substrate-binding residues. In the substrate binary complex, small movements of the side chain of Aspl02 and of Lys183 are sufficient to allow the amide oxygen to accept a hydrogen bond from the r-amino group of the lysine. In both the apo-enzyme and the Nbr8 ADP + complex, the lysine side chain interacts instead with a bound anion (sulphate 507 and pyrophosphate 506, respectively). It is tempting to associate this difference with the protonation state of the lysine even though the changes in coordinates are small relative to the resolution of the data sets. Neither the chemistry nor the positions in the binding site suggest any of the above residues except Lys183 are good candidates for catalytic groups in the reaction. Neither are they hydrogen-

6PGDH coenzyme specificity and mechanism Adams et al.

bonded in such a way that they would take part in any proton relay in the active site. Comparison with isocitrate dehydrogenase (IDH)

Of the well known NADP-dependent 03-ketoacid dehydrogenases, the three-dimensional structures are available for IDH and 6PGDH. Both malic enzyme and IDH require a divalent metal for activity. There is little similarity between the binding site for 6PG in 6PGDH and that for isocitrate in IDH [39] beyond the participation of both subunits of the dimer in both proteins. Although both enzyme reactions proceed via a general acid-general base mechanism, a magnesium ion stabilizes an enolate intermediate in the IDH reaction and the protein base and acid required are shared between subunits; the base has been identified as an aspartate (position 283 in the two-fold-related subunit in the E. coli enzyme) and the acid is either a lysine (position 230 in the two-fold-related subunit) or a tyrosine (position 160). Neither is there similarity in the three-dimensional structure: IDH does not have a Rossmann coenzyme fold; the coenzyme and substrate bind in a cleft between its two domains which share a mixed sheet. IDH is activated by dephosphorylation of a serine in the substrate-binding site. This serine forms a hydrogen bond to one of the carboxyls of isocitrate and the covalently bound phosphate prevents substrate binding. In contrast, in 6PGDH, phosphate is an inhibitor competitive with substrate, but it is not covalently bound to the enzyme; Ser128 makes a hydrogen bond to the 1-carboxyl of 6PG and is close enough to sulphate 507 in the apo-enzyme that they form hydrogen bonds to the same water (614). The phosphate ions have a similar role in the binding sites although there is covalent modification only in IDH. There is no sequence homology apparent between the two enzymes. Summary

The 2.3A and 2.5A refined structures of the dinucleotide complexes show that there are two ways of stabilizing the nicotinamide of the coenzyme in this protein, depending on its redox state. No enzyme conformational change involving main chain atoms is required or observed for either complex. The bound dinucleotides share with bound 2'AMP the same protein ligands to the adenine ring, 2'-phosphate and adenine ribose. Both coenzyme conformations are extended and both may represent stages in the enzyme mechanism: in the oxidized complex, the coenzyme is close to the orientation required for oxidation of the substrate 6PG and in the reduced complex, it is in a position where it may facilitate decarboxylation of the product or CO 2 release. Substrate specificity is achieved through binding of the phosphate moiety to Tyrl91, Arg287, Lys260 and Arg446 of the second subunit and of the carboxyl group to Ser128 and Glu190. Lys183 and an ordered water molecule are shown to be the most likely catalytic groups.

Biological implications The dehydrogenases of the pentose phosphate pathway contribute to its metabolic role by providing reduced nicotinamide adenine dinucleotide phosphate (NADPH) for synthesis; their NADP specificity is crucial. We have shown three contributions to NADP specificity for sheep 6phosphogluconate dehydrogenase (6PGDH). First, there is extensive interaction of the 2'-phosphate with Asn32, Arg33 and Thr34 which follow strand B of the dinucleotide-binding fold. Second, ordering Arg33 on binding NADP converts a hydrophobic surface depression into an adeninebinding pocket. Third, the alanine which substitutes for the second glycine of the dinucleotide binding 'fingerprint' obstructs hydrogen bonding between the bisphosphate and the first turn of helix xta. The binding energy derived from parts of the coenzyme other than the 2'-phosphate is thereby decreased, lowering the affinity for NAD. The arginine interaction is one general method of defining NADP specificity. Oxidative decarboxylation of 6-phosphogluconate (6PG) yields pentose sugars for nucleic acid synthesis. 6PGDH is specific for the 6-phosphate; a general base/acid mechanism has been proposed in which a protein base deprotonates the 6PG 3-hydroxyl. The structure of the co-crystallized 6PG-6PGDH complex defines the role of conserved residues in catalysis and specificity. Residues from the coenzyme and helical domains of one subunit are important in binding and catalysis, while close binding to the tail of the second monomer, which interacts closely with the first, enhances specificity for the 6-phosphate. The coenzyme and substrate binary complexes demonstrate that the oxidized coenzyme conformation, with a syn nicotinamide, would allow 6PG binding, whereas the reduced coenzyme competes with the 1-carboxyl of 6PG. A model for direct hydride transfer from the 3-hydrogen to the nicotinamide si- face is proposed in which small movements of the oxidized nicotinamide nucleotide have been made which require no protein movement. Lys183 is positioned to act as the base facilitating hydride transfer; it is deprotonated in presence of substrate but protonated in apo-enzyme crystals where two sulphate ions bind in the substrate site. Identification of the acid which catalyzes decarboxylation is less certain; the most likely candidate is a water hydrogen bonded to the main chain nitrogen of conserved Glyl30. The interactions of NADPH with the pro-

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Structure 1994, Vol 2 No 7

tein explain its competition with 6-phosphogluconate and are consistent with a possible role in decarboxylation or product release.

Materials and methods Enzyme isolation and preparation of complex crystals Enzyme was prepared as described previously [25]. Apo-enzyme crystals were grown from ammonium sulphate solution (50 mM) in mixed potassium phosphate (KPi) and buffered to pH6.5. Either the hanging drop method or a batch procedure was used. In hanging drop co-crystallization experiments, enzyme, precipitant (as above) and 6PG were in 20 ll drops; the well solution contained only precipitant. The conditions used are shown in Table 6, together with the relevant Michaelis constant (Km ) and dissociation constant (Kd). For soaking experiments, crystals were placed in the well solution or in a more concentrated ammonium sulphate solution than that from which they were grown. The soak time, buffer pH and concentration of coenzyme or analogue are given in Table 6, together with the relevant Kd, Km or inhibition constant (Ki). Concentrations in the order of 100 times the kinetic constant were used. For most complexes, coenzyme or analogue was soaked into apo-enzyme crystals. The first NADPH experiment used apo-enzyme crystals and the second 6PG co-crystals.

Coenzyme, coenzyme analogues and substrate NADPH, 2'AMP and 6PG were purchased from Sigma Chemical Co. Ltd. (Fancy Rd, Poole, Dorset, BH17 7NH, UK). We are grateful to J Neenan of Rochester Institute of Technology, NY, USA who prepared the sample of Nbr8 ADP + (according to the method of Abdallah et al. [24]) for these experiments.

X-ray data Different X-ray sources and/or detectors were used in the various experiments and data were collected to differing resolution. All complexes, including co-crystals, remained isomorphous with the apo-enzyme. The refined cell dimensions vary by no more than 0.6A ( < 1 %) from those of the apo-enzyme: a= 72.74(7)A, b = 148.40(11)A, c = 102.35(8) A (errors shown

Table 6. Conditions of preparation of complex crystals.

Kinetic parameters

Complex

Crystals

Soak conditions

Nbr8ADP+

apo-enzyme (hanging drop)

NbrADP 5mM KPi 50 mM, AS 56 %

NADPH 3.0 A

apo-enzyme (batch)

saturated, pH 6.5; 18 h NADPH 5 mM KPi 50 mM, AS 65 %

Kd in KPi = 5.7 jM 1171,

NADPH

enzyme grown in

saturated, pH6.5; 35min NADPH 5mM

in TEA = 0.45 M [1111 See above

2.5 A

presence of substrate (hanging drop) apo-enzyme (batch)

6PG 30 mM % KPi 50 mM, AS 52 saturated pH 7.0; 2 h 2'AMP 50 mM KPi 50 mM, AS 65 %/o saturated, pH 6.5; 2 h

Ki = 0.355 mM 181 + C wrt NADP NC wrt 6PG

Co-crystals

Co-crystals

drop solution:

well solution:

2'AMP

6PG

+

6PGDH: 5mgml-1 AS 54 %saturated, pH 6.5 (50mM KPi) 6PG 30mM AS 40 % saturated

K, = 9.0 lM [24]

K, in KP = 313 pM, in TEA = 23 M [111 Kd = 2 M [491

pH 6.5 (50 mM KPi) KPi = KH 2PO4 /K 2 HPO 4 buffer; AS = ammonium sulphate; TEA = triethanolamine; C = competitive; NC = non-competitive.

in parentheses). Initial data processing used the Xengen package [40] or MOSCO/MOSFLM [41,42], and further analysis used standard programs and procedures. Statistics for the data sets are given in Table 7.

Initial model building Difference maps against native data were calculated using the CCP4 Fast Fourier Transform program [SERC (UK) Collaborative Computer Project 4, Daresbury Laboratory, UK, 1986]. The protein phases used corresponded to the best current refinement of the apo-enzyme structure by simulated annealing (XPLOR) [43]; bound solvent was included in the phase calculation. Models were built into the difference density using the programs FRODO [44] and O [45] implemented on the Evans & Sutherland PS390 and ESV10 colour graphics systems. The small molecule coordinates for 6-phosphogluconate (tri-sodium salt) [46] were used as a starting model for bound substrate.

Table 7. Data collection parameters. Complex

8

+

Nbr ADP NADPH 2.5 A NADPH 3.0 A 2'AMP 6PG

Sourcea (wavelength)

Detector

dmax

nobsb (nind)

Residual Rm()C

Completeness o/o

F 2 4(crF) %0/0

mfidd

RA, CuKa (1.542 A) RA, CuKx (1.542 A) DL (0.88 A) DL (1.476 A) RA, CuKa (1.542 A)

multiwire (Siemens) image plate (MAR) film (CEA) film (CEA) image plate (MAR)

2.3 A

58176 (19 277) 46 279 (17995) 52125 (9438) 29 208 (8942) 44 279 (17 989)

6.2 %

77.2

0.11

6.7 %

92.2

8.3 %

88.0

67.3 (89.0-3 A) 86.5 (94.0-3A) 95.8

6.3 %

92.2

91.2

0.12

8.25 %

92.8

81.4

0.14

2.5 A 3.06 A 3.17 A 2.5 A

0.14 0.10

aRA refers to in-house rotating anode generator (run at 60 KV, 70 mA); DL refers to Daresbury Laboratory synchrotron radiation source. bnObs is the total number of observations; nind is the number of independent reflections. CRm(l) = (1hEi = 1,NI hi- < Ih > I)/(hNx < lh>) where h is the reflection index and is the mean of N equivalent intensity measurements (Ihi). dmfid is the mean fractional isomorphous difference (in F) from native.

6PGDH coenzyme specificity and mechanism Adams et al. Structure refinement The structures were improved by alternating cycles of refinement by simulated annealing and model building using omit/2F, F maps where the contribution from the substituent was omitted in the calculation of the model structure factor Fc . For the 2.5A NADPH and 6PG complexes, the protocol of Hodel et al. [47] was employed to reduce model bias further: residues within 5A of the coenzyme or substrate were omitted both from structure factor calculations and from molecular dynamics; those between 5 A and 10A from the coenzyme were restrained; and the remainder of the protein was refined in the normal way. Omit/2Fo-F c maps (for which the model contribution of residues within 5A of the coenzyme was not included) were calculated after this procedure. Bound waters were included in the NADPH, Nbr8 ADP + and 6PG complex structures when they were visible in the omit/2Fo-F maps with a density greater than 1.5c and made at least two potential hydrogen bond contacts at a distance of less than 4 A, either to the protein or to other bound waters; they were removed if their density fell significantly below cr in a map where they had been included in the phasing. The occupancy of the less well-defined waters, which had refined to high temperature factors, was set to 0.5. The occupancies of different portions of dinucleotides and of the sugar and the phosphate of 6PG were adjusted so that the temperature factors were not substantially greater than those of the atoms in the protein with which each made contact and there was a smooth variation throughout the substituent. All atoms were included in the final positional and temperature factor refinement of each complex. Final omit/2Fo-F c maps (illustrated in Fig. 3) were calculated using phases with the substituents omitted. Since the data for the 2'AMP complex extended only to 3.2A resolution, a different protocol was followed in refinement: the large domain of the protein was restrained and temperature factors were not refined. No attempt was made to adjust the water structure; waters close to the binding site were removed and other well bound waters left in the positions found in the (then current) refined apo-enzyme structure. Initial cycles of refinement of the 3.0A NADPH structure, using the same protocol as that for 2'AMP, allowed the nicotinamide ribose and nicotinamide to be built from omit/2FO-F c maps. The refinement was not pursued further after it was shown that the 2.5A NADPH data set corresponded to a binary reduced coenzyme complex with the same coenzyme conformation. X-ray amplitudes and phases for the native, coenzyme, coenzyme analogue and substrate bound structures and the derived atomic coordinates, have been deposited with the Brookhaven Protein Data Bank. Acknowledgements We are grateful to Professor LN Johnson for facilities and support. We wish to thank Dr. J Neenan who prepared

Nbr8 ADP+ for us, Dr. KCM Pelly who determined the conditions for the 2'AMP soaking experiment and collected the data, Dr. DC Harris who calculated the first 2'AMP difference map and Dr. DO'N Somers who determined the conditions for the 6PG co-crystallization. We acknowledge support from 'the MRC for a studentship (to CP). SG is funded by OCMS; MJA is Dorothy Hodgkin-EP Abraham Fellow of Somerville College and an associate member of OCMS.

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Received: 14 Mar 1994; revisions requested: 7 Apr 1994; revisions received: 16 May 1994. Accepted: 17 May 1994.