Beruflich Dokumente
Kultur Dokumente
*Corresponding author.
has been shown to act as a poor coenzyme in C utilis in the same monomer, one from the coenzyme domain
6PGDH with a Km 1000 times that for NADP + [16]. and two from the helical domain, and four contacts to
The reduced coenzyme binds more tightly than the oxi- bound water molecules.
dized species; for the sheep enzyme in phosphate, Kd The binding site for coenzyme has been reported
at pH 7.0 is 5.7 gM [17]. The affinity of both oxidized at low resolution [24]; it lies at the carboxy-termi-
and reduced coenzymes is enhanced 10-fold in TEA or nal end of the parallel -sheet. This paper describes
in acetate [11]. While most nucleoside phosphates in- crystallographic studies to define the binding of oxi-
hibit competitively with both coenzyme and substrate, dized and reduced coenzyme and of reduced substrate
2'AMP is competitive with NADP + and non-compet- at high resolution. Coenzyme or coenzyme analogue
itive with 6PG [18]. There is evidence that in some has been soaked into crystals and binding has been
circumstances NADPH has a role in decarboxylation of seen in difference electron density maps. In these ex-
the oxidized substrate [19]. periments, we have used NADPH, the active NADP+
Inorganic phosphate has been shown to stabilize analogue nicotinamide-8-bromo-adenine dinucleotide
6PGDH [20] and to inhibit competitively with both phosphate (Nbr8 ADP + ) [24] and 2'AMP, since it is the
coenzyme and substrate with inhibition constants (K only metabolite known to be competitive with coen-
i )
in the 4-8 mM range. Oxaloacetate, citrate, sulphate (Ki zyme alone. These molecules bind with very similar
8mM) and pyrophosphate also inhibit [18]. Of the conformations for the adenine, adenine ribose and 2'-
wide range of metabolic intermediates and analogues phosphate. The oxidized and reduced nicotinamide
investigated, 6-sulphogluconate, 6-phosphoallonate, 5- rings bind differently: all protein contacts to the oxi-
phosphoribonate, and 5-phosphoarabonate inhibit the dized analogue are from the coenzyme-binding do-
C utilis enzyme and are competitive with substrate main. The dihydronicotinamide makes contact with
while 6-phosphomannonate is a poor substrate [21]. residues in the helical domain which bind the 1-car-
Sheep liver 6PGDH is inhibited by fructose 1,6-bis- boxyl of 6PG in its binary complex. The structure of
phosphate (K i 0.07mM), by the 1- and 6-monophos- the co-crystallized substrate binary complex has shown
phates and by glucose 6-phosphate; all are competitive the general base to be the conserved lysine at position
to substrate only [18], with Ki values mostly in the 183. It has further shown that the residues responsible
millimolar range. The analogue 2-deoxy 6-phosphoglu- for the specificity of substrate binding come from both
conate [19] is a substrate which gives rise to an inter- the coenzyme and the helical domains of one subunit,
mediate keto-acid which is decarboxylated slowly. and from the tail of the second.
The two NADPH complexes are essentially identi- (505), which is tightly bound between subunits, dis-
cal if allowance is made for the difference in res- placed.
olution limit: an electron density map with terms For each dinucleotide, the occupancy which yielded
FNADPH(2.5A)-FNADPH(3.OA) and apo-enzyme phases appropriate thermal parameters (see Materials and
was flat (the largest peak or hole was < 1.5 % the height methods) was lower for the nicotinamide and nicoti-
of the 2'-phosphate peak in the 3.0A NADPH-apo-en- namide ribose than for the remainder of the coen-
zyme difference map). There was no density corre- zyme. Fractional occupancies were set at 0.5 and 0.8
sponding to substrate and it was concluded that the for Nbr 8 ADP + and at 0.5 and 0.7 for NADPH. (No
reduced coenzyme had displaced substrate from its precautions were taken to prevent NADPH oxidation
binding site. and the lower occupancy of the dihydronicotinamide
ring may reflect partial oxidation to NADP+, which
Density corresponding to the nicotinamide ribose and has a higher Kd. The short soak time used should
to both the oxidized and reduced nicotinamide rings diminish the extent of this problem.) The difference
appeared in their respective omit/2Fo-Fc maps during in fractional occupancies for the sugar of 6PG (0.75)
the course of the simulated annealing refinement. Sim- and for its 6-phosphate (1.0) are a consequence of
ilarly, the conformation of the 6PG molecule became incomplete substitution of the tightly-bound sulphate
apparent. In all these maps, the dinucleotide or sub- ion (505). More than 10 % of the sulphate can be
strate and solvent molecules within the binding cavity expected to remain bound. Using the solution values
were omitted from the phasing; for the NADPH and for Kd and K, Kd (6PG) = 2 M, Ki (SO42 -) = 8mM,
6PG complexes, they were also omitted from refine- [6PG] = 30mM, [S0 42 -] = 2.1M, the effective ratio
ment (see Materials and methods). In the oxidized (SO42- bound)/( 6 PGbound) = (2 x 10-6/8 x 10-3) x
coenzyme analogue, extended solvent was interpreted (2.1/30 x 10-3) = 0.13. Table 1 gives refinement statis-
as the competitive inhibitor pyrophosphate [18], which tics and Table 2 the parameters of the final models,
was used in eluting the enzyme from an affinity column with the coordinate errors derived from the change of
[25]. In Fig. 3, the coenzyme and substrate coordinates residual with resolution [26].
are superimposed on the final omit/2Fc-Fc maps for Coenzyme, coenzyme analogue and substrate
the 2.5A NADPH, Nbr 8ADP + and 6PG complexes. For conformations
each complex, the substituent and solvent within the The conformations of the adenine, adenine ribose and
binding site were omitted from the phase calculation. 2'-phosphate are very similar in the three structures.
In no coenzyme bound structure was the sulphate ion Although the dinucleotides in both complexes are ex-
654 Structure 1994, Vol 2 No 7
tended, the nicotinamide ribose and nicotinamide of plex are given in Table 3, for all residues and for those
the oxidized and reduced dinucleotides have differ- within 10A of the substituent. There is little protein
ent conformations. The greater extension of the re- movement required to accommodate the coenzyme
duced coenzyme can be seen in Fig. 4. The adenine other than the ordering of the side chain of Arg33 and a
ring is anti for all three complexes. While the plane small movement of some of the residues in two loops:
of the nicotinamide ring is close to that of the nicoti- 73-78 (3D-ad) and 130-133 (PF-a-f). The pattern of
namide ribose in both dinucleotide complexes, the main-chain temperature factors is similar to that of the
conformation is essentially syn in the Nbr8ADP + com- apo-enzyme. Many of the well-bound waters are com-
plex. The adenine ribose is C2'-endo (C3'-exo) in all mon to apo-enzyme and complexes; details of the num-
three complexes; the nicotinamide ribose is C3'-endo ber and proportion in common are given in Table 3.
in the NbrsADP + complex and close to C2'-endo in The coenzymes replace ordered waters: 11 waters and
the NADPH complex. a sulphate are displaced by the nicotinamide nucleotide
The conformation of the bound 6PG is shown in Fig. 5, of NADPH, 2 waters by the adenine nucleotide. Twelve
where it is compared with the small molecule structure of these waters, and two extra ones, are displaced by
(tri-sodium salt). The root mean square (rms) differ- Nbr8 ADP +; the sulphate and one remaining water are
ence in the superimposed coordinates is 1.49k The replaced by the pyrophosphate. On binding coenzyme,
most striking difference between the conformations is the solvent accessibilities of several residues in the
the orientation of the 1-carboxyl with respect to the binding site are decreased. These residues are: Alal 1,
carbon backbone. Metl3, Arg33, Lys37, Leu73, Val74, Lys75 and Ala79.
The hydrophobic residues amongst these are more ex-
Binding sites posed than the average of their types in the apo-en-
The mean differences of positional parameters be- zyme and contribute towards an exposed hydrophobic
tween the apo-enzyme and the protein in each com- patch.
6PGDH coenzyme specificity and mechanism Adams et al. 655
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.
and is not responsible for any other perturbation of the bonds are made by the y-hydroxyl of Thr34 and both
enzyme structure. the oxygen and nitrogen of the amide of Asn32.
In both dinucleotide complexes, the adenine ribose
There are potential hydrogen bond interactions to the and bis-phosphate make van der Waals contact with
2'-phosphate in all complexes from the side chains of three residues of the tight 3A-aa turn (Gly9, LeulO
three residues in the loop between PB3and cab: Asn32, and Alall) and with Val74 and Lys75 of the 3D-ctd
Arg33 and Thr34. The interactions involve at least three turn. The 3'-hydroxyl of the adenine ribose is within
of the phosphate oxygens with often more than one hydrogen-bonding distance of the main chain nitrogen
hydrogen bond being possible to each. The hydrogen of LeulO and the main chain nitrogen of Lys75 can
6PGDH coenzyme specificity and mechanism Adams et al. 657
Coenzyme Mean positional difference Waters in common Residues within 10A with large
Residues within 10A All residues with apo-enzymea positional differencesb
mc sc mc sc mc sc
Nbr8 ADP + 0.249 A 0.413 A 0.236 A 0.566 A 126 (30 %) 74, 75, 76, 77, 78 33, 75, (260)
368 atoms 332 atoms (130), (131), 132,
259, 260
NADPH 0.230A 0.535 A 0.188A 0.488A 130 (31%) (74), (75), (131), 33, (73), (74),
368 atoms 332 atoms 132, (133), (260), 75, (260)
(262), (450)
6PG 0.219A 0.508A o.199A 0.492 A 161 (40 %) (75), (132), (73), 75
272 atoms 236 atoms (447), (448)
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 hydrogen bonds Oy of Ser128 at the end of PF, NE2 of
ribose. In all complexes, 061 of Asn32 also interacts His186 and 061 of Asnl87; both these last residues are
with this 3'-hydroxyl group. in the first helix of the helical domain (ah) and are con-
served for all known sequences. Additional conserved
Nicotinamide nucleotide contacts residues from this helix (Lys183 and Glu190) make van
The remaining interactions differ between the oxidized der Waals contact, primarily with the amide function.
and reduced coenzyme complexes. In the complex
with the oxidized analogue, Lys75 extends to follow the Protein-substrate contacts
shape of the nicotinamide ribose and van der Waals The inter-subunit nature of the substrate site is immedi-
interactions are also made with the loop between PE ately apparent, as is the very large proportion of totally
and ae (residues 100-104). AsplO02 in this turn makes conserved residues within 4A of 6PG. The substrate
van der Waals contact with the ribose and is com- makes hydrogen bonds to residues in the F--af loop of
pletely conserved in all seven species. Almost all in- the coenzyme domain and to residues in three regions
teractions with the nicotinamide are made by the con- of the helical domain of one subunit: one face of the
served Metl3; the thioether is above the nicotinamide helix ah, the end of the long cj-ak loop and Arg287
ring. The only additional direct hydrogen bonds to pro- in al. Hydrogen bonds are also made to residues in
tein from the oxidized coenzyme complex are made the tail of the second subunit of the dimer, in cts and
by the nicotinamide amide: the oxygen with the main in the turn immediately following it. The interactions
chain of Metl3 and the -NH2 group with the carboxyl of to the 6-phosphate of 6PG are the same as those to
Glu131 at the beginning of af. The pyrophosphate ion, sulphate 505 in the apo-enzyme. There is no direct
which replaces sulphate 507 in this complex, interacts match between sulphate 507 and the carboxyl of 6PG;
with Ns of Lys183 (in h), the amide of AsplO02, the the carboxyl interactions match the contacts to water
main chain nitrogen groups of the residues of the tight 614 of the apo-enzyme most closely. This water forms
,F-af turn (Gly129 and Gly130), and with two waters a hydrogen bond to sulphate 507, as does water 613
(614 and 886) which are present in the apo-enzyme which is also displaced by the substrate. There is a
structure. The charge of the pyridinium ring is coun- considerable overlap between residues interacting with
terbalanced by the bound pyrophosphate ion. the carboxylate and those interacting with the reduced
nicotinamide. His186 is within 4A of the carboxyl and,
In the NADPH complex, the nicotinamide ribose, in- if it is protonated, will serve to balance the negative
stead of interacting with Lys75, contacts N82 of Asnl02 charge.
in the loop between E and ae. The bis-phosphate
is hydrogen-bonded through water 589 to the main The carboxyl group of the substrate bonds to Oy of
chain nitrogen of both Alall and Gly14 (residues at Serl28 and to 0O1 of Glul90; the latter had no close
the beginning of ca) and to the carbonyl oxygen of neighbour in the apo-enzyme. The second oxygen of
Leu73 (in the fID-cd loop), through water 692 to the the carboxyl group of 190 hydrogen bonds the con-
main chain nitrogen of Metl3 and through a third water served water 528 in all complexes and in the apo-
(516) to the main chain nitrogen of AsnlO02. All three enzyme. This water makes two additional hydrogen
waters are also present in the apo-enzyme structure. bonds: to Arg287 Nq2 in both structures; and to 02
Instead of Metl3, residues from the PF-af turn, Val127, of the 6-phosphate of 6PG or to the equivalent oxy-
and the totally conserved Serl28, Gly129 and Glyl30 gen (03) of sulphate 505. The 2-hydroxyl (08) makes
interact with the nicotinamide. The nicotinamide amide direct hydrogen bonds to two waters; water 1232 in
658 Structure 1994, Vol 2 No 7
turn contacts the main chain nitrogen of Gly130 of the nine ring has also been observed when nicotinamide-
OF-af turn; water 1109 has no other ordered interac- 8-bromo-adenine dinucleotide is bound to glyceralde-
tions. In contrast, the 6PG 3-hydroxyl makes two direct hyde 3-phosphate dehydrogenase (GAPDH) [28] and
hydrogen bonds to protein: to N of Lys183 and to in other enzyme complexes. The 6PGDH coenzyme
N62 of Asn187. While the C4 and C5 hydroxyls do not site, in common with those of other dehydrogenases,
make good hydrogen bonds in the binary complex, is thus specific for the anti conformer. The close su-
His452 of the second subunit approaches them and perposition of 2'AMP with the comparable fragments
a small change in conformation would allow at least of the dinucleotides is consistent with inhibition com-
one hydrogen bond to be made. These hydroxyls are petitive only with coenzyme.
constrained by the tight interactions of the 6-phosphate
and the oxygens of C1, C2 and C3. Specificity of protein for NADP; importance of Arg33
Discrimination between NAD and NADP binding re-
quires that the energy of the 2'-phosphate-protein
interaction is a large proportion of the total bind-
Discussion ing energy. This may be achieved by a large net 2'-
Comparison of coenzyme conformations in binary phosphate-protein binding energy, a small net binding
complexes energy of the remainder of the coenzyme, or both.
The experiments described above show that 2'AMP,
the reduced coenzyme and an active oxidized coen- The 2'-phosphate dominates in the hydrogen bond in-
zyme analogue, modified at the adenine, will bind to teractions made directly to the protein (shown in Table
apo-6PGDH in the crystal without invoking any large 4), taking part in 7 of 14 direct interactions of NADPH,
conformational change of the enzyme. Both coenzymes 6 of 11 interactions of Nbr 8ADP + and 5 of 7 inter-
bind in an open conformation, but the conformations actions of 2'AMP. Of the three consecutive residues
beyond the bis-phosphate are distinct. The reduced in the turn between B and acb making these hydro-
coenzyme in the 6PGDH complex is as extended as is gen bonds, Asn32 is conserved in all seven known se-
the coenzyme in dihydrofolate reductase (DHFR). The quences, Arg33 is replaced by tyrosine in one sequence
distance between C6 of the adenine ring and C2 of the (that of B. subtilis) and Thr34 is replaced by serine
nicotinamide ring is usually taken as a measure of this in both the E coli and S. typhimurium sequences.
extension. For most bound coenzymes, the distance is Identical hydrogen-bonding potential is thus retained
between 14A and 15A; for NADPH bound to DHFR, it at residues 32 and 34 and a planar residue with ca-
is 17.1A [23]. The distance for Nbr8 ADP + bound to pacity to act as a hydrogen bond donor at 33. The
6PGDH is 15.1A; that for bound NADPH is 17.8k dominance of the 2'-phosphate interaction must make
a major contribution to the NADP + specificity of this
The conformations of 2'AMP and the adenine mononu- enzyme. The position of Asn32 in the turn between
cleotide moieties of the two dinucleotides are closely ,B and ab is the same as that of the aspartate which
similar. The anti conformation of the adenine ring hydrogen bonds the adenine ribose in NAD-dependent
is anticipated for the unsubstituted adenine moieties, dehydrogenases [29]. In 6PGDH, acb is oriented and
but for the Nbr8 ADP+ complex it contrasts with the positioned so that its amino terminus is approximately
syn conformation preferred in free 8-bromo-adeno- 5A from the 2'-phosphate and the helix dipole may
sine [27]. The anticonformation of the 8-bromo-ade- contribute to stabilizing the negative charge.
660 Structure 1994, Vol 2 No 7
Coenzyme NbrSADP+ NADPH 2'AMP 6PG region 6PG complex Inorganic ion Apo-enzyme
moiety
c d
Bis-phosphate O1" O Wat692 6-phosphate S0 4505 01-0 Wat886
02"-0 Wat589 03-O I Tyr191* 02-OT0 Tyr191*
05(R)-O Wat516b 03 NH Lys260* 02 NH Lys260'
*b
03-Oy1 Thr262 02-0 Wat953
Nicotinamide 04-N2 Asn102* 02 Nll1 Arg287* 03-NTrl Arg287-
ribose 02-Nfll Arg446*# 03 Nill Arg446*#
02-0 Wat528 03-0 Wat528
Nicotinamide 07-NH Met13* 07-Oy Ser128' 01-Nfll Arg446*# 04-Nfl1 Arg446*#
N70-OE2 Glu131' 07-Nc2 His186'
N7-061 Asn187' Water Water
neighbours neighbours
Pyrophosphate 01'-061 Asn102* Wat528 0-02 Glu190* Wat528 0-062 Glu190
01'- N2 Asn102* O N13 Arg287* O-NTl2 Arg287
02'-Nr Lys183' (0-02 6PG) (0-03 S0 4505)
02'-0 Wat614 Wat1109 (0-08 6PG) Wat613 O0NH Gly129
c
04'-0 Wat886c Wat1232 O-NH Glyl130 O NH Gly130*
02" 0 Val127 (0-08 6PG) (0-01 504507)
02"-Nr Lys183' 0 0 Wat614
02"0 Wat614 (0-04 504507)
04"-NH Gly129* Wat614 O-Oy Ser128*
04" NH Gly130' 0 0 Wat613
O-NE2 His186
Waters 0614-OY Ser128' 0516-NH Asn102* (0-01 S0 4 507)
0614-01 Glu190' 0589-NH Alall Wat699 0-0 Va1127
0886-NE2 His452# 0589-NH Gly14* (0-04 504507)
0886-04 504505 0589-0 Leu73 Wat886 0-No2 His452'
0589-0 Gly9'(? (0-01 504 505)
)
0692 NH Met13 Wat953 (0-03 50 4 50 7 b
O-NH Thr262'
aContacts with donor-acceptor distances 3.3A or less are defined as potential O-Oyl Thr262-
hydrogen bonds. Longer distances (b = 3.4 A; c = 3.5 A) are indicated in the table. 0-02 S0 4505
Less satisfactory angles are indicated by (?). Totally conserved residues are indi-
cated by (), two-fold related subunit by (#), water by Wat. 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 satis-
factory angle. do1, 02, 03, 04 of sulphate 505 correspond to 04, 03, 02, 01
of 6PG 6-phosphate.
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 bind- mation of the arginine in the apo-enzyme makes van
ing coenzyme and, as well as binding the 2'-phosphate der Waals contacts with some of the residues which
and providing a charge balance, it is responsible for form the hydrophobic contacts to adenine. A similar
all the contacts to one face of the adenine ring; the adenine-arginine interaction has been observed in the
guanidinium group and the adenine ring are stacked NADP-dependent glutathione reductase (GR) [30] and
and approximately coplanar. The depth of the adenine arginine is involved in stabilizing the adenine and 2'-
cleft in sheep 6PGDH is limited by the phenylalanine phosphate of NADP in avian DHFR [31] and in cata-
side chain of residue 83 in ctb. This restricts the pos- lase [32]. This interaction may be identified as one of
sibilities for the adenine site and contributes towards the common solutions to the problem of enhancing
placing the adenine nearer the surface of the enzyme specificity for NADP relative to NAD. It is likely that the
than it is in the NAD dehydrogenases. Fig. 8 illustrates driving force for ordering the arginine in 6PGDH is the
the difference the ordering of Arg33 makes to the ade- phosphate interaction and that, in the absence of the
nine pocket, showing that the most favoured confor- 2'-phosphate, there is no adenine cleft.
6PGDH coenzyme specificity and mechanism Adams et al. 661
,H:B .
A~ ,H:B
B.-
:
-O H 2
B:H' H- :-OH
2
H2 OP0 3 -
C02 -- 1 c02
IH 2 OH :B
B H--- NADPH
N
NADPH
-OH
-OH Fig. 14. Steps of the general base-acid
2' mechanism for 6PG oxidative decar-
H2 OPO3
boxylation by 6PGDH (after Berdis and
Cook [21]).
no evidence for a kinetically important conformational There are two conserved histidines in the binding site:
change, the possibility of a local structural rearrange- residue 186 and residue 452 of the two-fold-related
ment, particularly one involving coenzyme, cannot be subunit. Neither histidine is in a suitable position to act
ruled out. The structure of a product complex or of as base (or as acid) in the mechanism. His186 interacts
a complex with a ribulose 5-phosphate analogue is re- with Ser128 which stabilizes the 1-carboxylate of 6PG;
quired before the second acidic group can be identified NE2 of the histidine is 3.6 A from one of the carboxyl
definitively, but probable candidates are apparent in oxygens. If His186 is protonated it balances the car-
the 6PG binary complex. boxylate charge; if neutral, it would have only a sec-
ondary role in substrate binding. The distance between
Water 1232, one of the two waters which form hydro-
either ring nitrogen and the 3-hydroxyl or the 2-carbon
gen bonds to the 2-OH of 6PG, is a possible general
of the substrate is more than 5.5 A and His186 could
acid. This water contacts, and is probably hydrogen-
only act as base or acid if there were a significant con-
bonded to, the hydrogen of the main chain amide
formational change. His452 from the two-fold-related
group of the conserved Glyl30; hydrogen bond dona-
subunit makes its closest approach to the C4 and C5
tion from the protein would contribute to the water's
hydroxyls, contributing towards specificity of the site; it
enhanced acidity. An alternative hypothesis involves the
is more than 6A from the 3-hydroxyl or 2-carbon and
conserved glutamic acid at position 190 as a general
some 5.5A from the modelled nicotinamide position.
acid. The carboxyl group of this residue is hydrogen
The several chemical modifications which appeared to
bonded to the carboxyl of 6PG; one of these carboxyls
indicate that histidine was essential are likely to have
must be protonated. Diffusion away of the product CO 2
obstructed the substrate-binding site.
removes the requirement for the glutamate to be pro-
tonated and it could act as acid in the final tautomeriza-
tion. The substrate binary complex structure, however, The side chains of AsnlO2, Ser128, Tyrl91 and Thr261
argues against direct involvement of the glutamate since all contribute to binding specificity by direct interac-
the carboxyl oxygens are more than 5A from C2 of tions with substrate or by forming hydrogen bonds
the substrate and any closer approach would be at the to substrate-binding residues. In the substrate binary
expense of the interaction with water 528. The more complex, small movements of the side chain of Aspl02
likely role for a glutamate with a raised pKa is that of en- and of Lys183 are sufficient to allow the amide oxygen
hancing binding specificity for the substrate carboxyl. to accept a hydrogen bond from the r-amino group of
the lysine. In both the apo-enzyme and the Nbr8 ADP +
Role of some conserved residues in binding and catalysis complex, the lysine side chain interacts instead with
The phosphate group of 6PG makes close contacts a bound anion (sulphate 507 and pyrophosphate 506,
with the two conserved arginines in the binding site: respectively). It is tempting to associate this difference
residue 287 and residue 446 of the second subunit with the protonation state of the lysine even though the
of the dimer. These arginines balance the phosphate changes in coordinates are small relative to the reso-
charge and provide specificity. The network of hydro- lution of the data sets. Neither the chemistry nor the
gen bonds from Arg287 through water 528 to Glu190 positions in the binding site suggest any of the above
and the 1-carboxyl of 6PG is important in defining the residues except Lys183 are good candidates for cat-
bound 6PG conformation. alytic groups in the reaction. Neither are they hydrogen-
6PGDH coenzyme specificity and mechanism Adams et al. 665
+
Nbr8ADP+ apo-enzyme NbrADP 5mM K, = 9.0 lM [24]
(hanging drop) KPi 50 mM, AS 56 %
saturated, pH 6.5; 18 h
NADPH apo-enzyme NADPH 5 mM Kd in KPi =
Materials and methods
3.0 A (batch) KPi 50 mM, AS 65 % 5.7 jM 1171,
Enzyme isolation and preparation of complex crystals saturated, pH6.5; 35min in TEA = 0.45 M [1111
Enzyme was prepared as described previously [25]. Apo-enzyme NADPH enzyme grown in NADPH 5mM See above
crystals were grown from ammonium sulphate solution (50 mM) 2.5 A presence of 6PG 30 mM
in mixed potassium phosphate (KPi) and buffered to pH6.5. substrate KPi 50 mM, AS 52 %
Either the hanging drop method or a batch procedure was used. (hanging drop) saturated pH 7.0; 2 h
2'AMP apo-enzyme 2'AMP 50 mM Ki = 0.355 mM 181
In hanging drop co-crystallization experiments, enzyme, precip- +
(batch) KPi 50 mM, AS 65 %/o C wrt NADP
itant (as above) and 6PG were in 20 ll drops; the well solution
saturated, pH 6.5; 2 h NC wrt 6PG
contained only precipitant. The conditions used are shown in 6PG Co-crystals Co-crystals
Table 6, together with the relevant Michaelis constant (Km ) and drop solution: well solution: K, in KP = 313 pM,
dissociation constant (Kd). 6PGDH: 5mgml-1 AS 54 %saturated, in TEA = 23 M [111
6PG 30mM pH 6.5 (50mM KPi) Kd = 2 M [491
For soaking experiments, crystals were placed in the well so- AS 40 % saturated
lution or in a more concentrated ammonium sulphate solution pH 6.5 (50 mM KPi)
than that from which they were grown. The soak time, buffer pH
and concentration of coenzyme or analogue are given in Table
2 HPO 4 buffer; AS = ammonium sulphate; TEA = triethanolamine;
KPi = KH 2PO4 /K
6, together with the relevant Kd, Km or inhibition constant (Ki). C = competitive; NC = non-competitive.
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 in parentheses). Initial data processing used the Xengen pack-
used apo-enzyme crystals and the second 6PG co-crystals. age [40] or MOSCO/MOSFLM [41,42], and further analysis used
standard programs and procedures. Statistics for the data sets
Coenzyme, coenzyme analogues and substrate are given in Table 7.
NADPH, 2'AMP and 6PG were purchased from Sigma Chemi-
cal Co. Ltd. (Fancy Rd, Poole, Dorset, BH17 7NH, UK). We are Initial model building
grateful to J Neenan of Rochester Institute of Technology, NY, Difference maps against native data were calculated using the
USA who prepared the sample of Nbr8 ADP + (according to the CCP4 Fast Fourier Transform program [SERC (UK) Collabora-
method of Abdallah et al. [24]) for these experiments. tive Computer Project 4, Daresbury Laboratory, UK, 1986]. The
protein phases used corresponded to the best current refine-
X-ray data ment of the apo-enzyme structure by simulated annealing (X-
Different X-ray sources and/or detectors were used in the var- PLOR) [43]; bound solvent was included in the phase calcu-
ious experiments and data were collected to differing reso- lation. Models were built into the difference density using the
lution. All complexes, including co-crystals, remained isomor- programs FRODO [44] and O [45] implemented on the Evans &
phous with the apo-enzyme. The refined cell dimensions vary Sutherland PS390 and ESV10 colour graphics systems. The small
by no more than 0.6A ( < 1 %) from those of the apo-enzyme: molecule coordinates for 6-phosphogluconate (tri-sodium salt)
a= 72.74(7)A, b = 148.40(11)A, c = 102.35(8) A (errors shown [46] were used as a starting model for bound substrate.
8 +
Nbr ADP RA, CuKa multiwire 2.3 A 58176 6.2 % 77.2 67.3 0.11
(1.542 A) (Siemens) (19 277) (89.0-3 A)
NADPH RA, CuKx image plate 2.5 A 46 279 6.7 % 92.2 86.5 0.14
2.5 A (1.542 A) (MAR) (17995) (94.0-3A)
NADPH DL film 3.06 A 52125 8.3 % 88.0 95.8 0.10
3.0 A (0.88 A) (CEA) (9438)
2'AMP DL film 3.17 A 29 208 6.3 % 92.2 91.2 0.12
(1.476 A) (CEA) (8942)
6PG RA, CuKa image plate 2.5 A 44 279 8.25 % 92.8 81.4 0.14
(1.542 A) (MAR) (17 989)
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 <lh> 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. 667
Structure refinement 4. Nasoff, M.S., Baker, H.V. &Wolf, R.E. Jr. (1984). DNA sequence
The structures were improved by alternating cycles of refinement of the Escherichia coli gene, gnd for 6-phosphogluconate de-
by simulated annealing and model building using omit/2F, F hydrogenase. Gene 27, 253-264.
5. Reeves, P. & Stevenson, G. (1989). Cloning and nucleotide se-
maps where the contribution from the substituent was omit-
quence of the Salmonella typhimurium LT2 gnd gene and its
ted in the calculation of the model structure factor Fc . For the homology with the corresponding sequence of Escherichia coli
2.5A NADPH and 6PG complexes, the protocol of Hodel et K12. Mol Gen. Genet. 217, 182-184.
al. [47] was employed to reduce model bias further: residues 6. Broedel, S.E. & Wolf, R.E. Jr. (1990). Genetic tagging, cloning,
within 5A of the coenzyme or substrate were omitted both and DNA sequence of the Synechococcus sp. strain PCC 7942
from structure factor calculations and from molecular dynamics; gene (gnd) encoding 6-phosphogluconate dehydrogenase. J Bac-
those between 5 A and 10A from the coenzyme were restrained; teriol. 172, 4023-4031.
and the remainder of the protein was refined in the normal 7. Fujita, Y., Fujita, T., Miwa, Y., Nihashi, J.-I. & Aratani, Y. (1986).
way. Omit/2Fo-F c maps (for which the model contribution of Organization and transcription of the gluconate operon, gnt, of
residues within 5A of the coenzyme was not included) were Bacillus subtilis. J Biol Chem. 261, 13744-13753.
8. Scott, MJ. & Lucchesi, J.C. (1991). Structure and expression of
calculated after this procedure. Bound waters were included in
the Drosophila melanogastergene encoding 6-phosphogluconate
the NADPH, Nbr8 ADP + and 6PG complex structures when they dehydrogenase. Gene 109, 177-183.
were visible in the omit/2Fo-F maps with a density greater than 9. Harbitz, 1., et al., & Davies, W. (1990). Isolation, characterization
1.5c and made at least two potential hydrogen bond contacts and chromosomal assignment of a partial cDNA for porcine 6-
at a distance of less than 4 A, either to the protein or to other phosphogluconate dehydrogenase. Hereditas 112, 83-88.
bound waters; they were removed if their density fell significantly 10. Reizer, A., Deutscher, J., Saier, M.H. Jr. &Reizer, J. (1991). Anal-
below cr in a map where they had been included in the phasing. ysis of the gluconate (gnt) operon of Bacillus subtilis Mol.
The occupancy of the less well-defined waters, which had refined Microbiol. 5, 1081-1089.
to high temperature factors, was set to 0.5. The occupancies 11. Topham, C.M., Matthews, B. & Dalziel, K. (1986). Kinetic studies
of different portions of dinucleotides and of the sugar and the of 6-phosphogluconate dehydrogenase from sheep liver. Eur. J
Biochem. 156, 555-567.
phosphate of 6PG were adjusted so that the temperature factors
12. Berdis, A.J. &Cook, P.F. (1993). Overall kinetic mechanism of 6-
were not substantially greater than those of the atoms in the phosphogluconate dehydrogenase from Candida utilis Biochem-
protein with which each made contact and there was a smooth istry 32, 2036-2040.
variation throughout the substituent. All atoms were included in 13. Villet, R.H. & Dalziel, K. (1972). Studies of 6-phosphogluconate
the final positional and temperature factor refinement of each dehydrogenase from sheep liver: 2. Kinetics of the oxidative-de-
complex. Final omit/2Fo-F c maps (illustrated in Fig. 3) were carboxylation reaction, coenzyme binding and analyses for metal.
calculated using phases with the substituents omitted. Eur. J Biochem. 27, 251-258.
14. Rendina, A.R., Hermes, J.D. & Cleland, W.W. (1984). Use of mul-
Since the data for the 2'AMP complex extended only to 3.2A
tiple isotope effects to study the mechanism of 6-phosphoglu-
resolution, a different protocol was followed in refinement: the conate dehydrogenase. Biochemistry 23, 6257-6262.
large domain of the protein was restrained and temperature 15. Topham, C.M. & Dalziel, K. (1986). The chemical mechanism
factors were not refined. No attempt was made to adjust the of sheep liver 6-phosphogluconate dehydrogenase. A Schiff-base
water structure; waters close to the binding site were removed intermediate is not involved. Biochem. J. 234, 671-677.
and other well bound waters left in the positions found in the 16. Berdis, A.J. & Cook, P.F. (1993). The 2'-phosphate of NADP is
(then current) refined apo-enzyme structure. Initial cycles of re- critical for optimum productive binding to 6-phosphogluconate
finement of the 3.0A NADPH structure, using the same protocol dehydrogenase from Candida utilis Arch. Biochem. Biophys 305,
as that for 2'AMP, allowed the nicotinamide ribose and nicoti- 551-558.
namide to be built from omit/2FO-F c maps. The refinement was 17. Silverberg, M. & Dalziel, K. (1975). Fluorescence studies of
coenzyme binding to 6-phosphogluconate dehydrogenase. Arch.
not pursued further after it was shown that the 2.5A NADPH
Biochem. Biophys 168, 646-651.
data set corresponded to a binary reduced coenzyme complex 18. Dyson, J.E.D. & D'Orazio, R.E. (1973). Sheep liver 6-phosphoglu-
with the same coenzyme conformation. conate dehydrogenase: inhibition by nucleoside phosphates and
X-ray amplitudes and phases for the native, coenzyme, coen- by other metabolic intermediates. J. BioL Chem. 248, 5428-5435.
zyme analogue and substrate bound structures and the derived 19. Hanau, S., Dallochio, F. & Rippa, M. (1992). NADPH activates a
atomic coordinates, have been deposited with the Brookhaven decarboxylation reaction catalysed by lamb liver 6-phosphoglu-
Protein Data Bank. conate dehydrogenase. Biochim. Biophys Acta 1122, 273-277.
20. Rippa, M., Signorini, M. & Bellini, T. (1981). The effect of inor-
Acknowledgements We are grateful to Professor LN Johnson for fa- ganic phosphate on the stability of some enzymes. Biochem. J
cilities and support. We wish to thank Dr. J Neenan who prepared 197, 747-749.
Nbr8 ADP+ for us, Dr. KCM Pelly who determined the conditions for 21. Berdis, AJ. & Cook, P.F. (1993). Chemical mechanism of 6-
the 2'AMP soaking experiment and collected the data, Dr. DC Harris phosphogluconate dehydrogenase from Candida utilis from pH
who calculated the first 2'AMP difference map and Dr. DO'N Somers studies. Biochemistry 32, 2041-2046.
who determined the conditions for the 6PG co-crystallization. We ac- 22. Adams, MJ., Gover, S., Leaback, R., Phillips, C. & Somers, D.O'N.
knowledge support from 'the MRC for a studentship (to CP). SG is (1991). The structure of 6-phosphogluconate dehydrogenase re-
funded by OCMS; MJA is Dorothy Hodgkin-EP Abraham Fellow of fined at 2.5A resolution. Acta Crystallogr. B 47, 817-820.
Somerville College and an associate member of OCMS. 23. Grau, U.M. (1982). Structural interactions with enzymes. In The
Pyridine Nucleotide Coenzymes. (Everse, J., Anderson, B. &You,
K.-S., eds), pp. 135-187, Academic Press Inc., New York & Lon-
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1.3. Protein Engineering Department, Genex Corporation, Mary- Received: 14 Mar 1994; revisions requested: 7 Apr 1994;
land, USA revisions received: 16 May 1994. Accepted: 17 May 1994.