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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 of 6-phosphogluconate is hydrogen bonded to Nr of


phosphate (NADP)-dependent oxidative decarboxylase, Lys183 and the 3-hydrogen points towards the oxidized
6-phosphogluconate dehydrogenase, is a major source nicotinamide. The 6-phosphate replaces a tightly bound
of reduced coenzyme for synthesis. Enzymes later in the sulphate in the apo-enzyme.
pentose phosphate pathway convert the reaction prod- Conclusions: NADP specificity is achieved primarily
uct, ribulose 5-phosphate, to ribose 5-phosphate. Crys- by Arg33 which binds the 2'-phosphate but, in its ab-
tallographic study of complexes with coenzyme and sub- sence, obscures the adenine pocket. The bound oxi-
strate explain the NADP dependence which determines
the enzyme's metabolic role and support the proposed dized nicotinamide is syn; hydride transfer from bound
general base-general acid mechanism. substrate to the nicotinamide si- face is achieved with a
Results: The refined structures of binary coenzyme/ small movement of the nicotinamide nucleotide. Lys183
analogue complexes show that Arg33 is ordered by may act as general base. A water bound to Glyl30 in the
binding the 2'-phosphate, and provides one face of the coenzyme domain is the most likely acid required in de-
adenine site. The nicotinamide, while less tightly bound, carboxylation. The dihydronicotinamide ring of NADPH
is more extended when reduced than when oxidized. All competes for ligands with the 1-carboxyl of 6-phospho-
substrate binding residues are conserved; the 3-hydroxyl gluconate.

Structure 15 July 1994, 2:651-668


Key words: enzyme mechanism, NADP/NADPH binding, 6-phosphogluconate dehydrogenase, substrate binding

Introduction phosphate buffer, where there is evidence that NADP +


The pentose phosphate pathway enzyme 6-phospho- may bind before 6PG, the dissociation constant (Kd)
gluconate dehydrogenase (6PGDH; EC 1.1.1.44) oxida- for NADP+ is 26~ M [13] and the Michaelis constant
tively decarboxylates 6-phosphogluconate (6PG) to (Km ) for 6PG is 313 M. In triethanolamine (TEA) at
give ribulose 5-phosphate (Ru5P). The enzyme is pH 7.0, the dominant path is through a complex of
dimeric and NADP-dependent for almost all species enzyme with 6PG, and the Km for 6PG was calculated as
[1]. Seven complete primary sequences have been de- 23 [M. Product release is ordered in either buffer with
termined (for sheep [2]; Trypanosoma brucei [3]; carbon dioxide leaving first, followed by Ru5P. 6PGDH
Escherichia coli [4]; Salmonella typhimurium [5]; oxidizes the 3-hydroxyl of 6PG and decarboxylates the
Synechococcus [6]; Bacillus subtilis [7]; Drosophila resulting 3-keto,6-phosphogluconate. Multiple isotope
melanogaster [8]) and two further partial sequences effects have been interpreted in terms of a sequen-
(pig [9,10]; mouse: S Hoffmann, personal communica- tial mechanism [14]; however, a recent investigation
tion). The subunit contains between 468 (E. coli) and under a wider range of conditions suggests that an
482 residues (sheep), 88 of which are conserved in all asynchronous concerted oxidative decarboxylation af-
sequences reported so far. ter formation of a C-3 alkoxide cannot be ruled out (P
Cook, personal communication). An acid-base mecha-
The kinetic mechanism has been studied most in- nism has been proposed; involvement of a Schiff base
tensely for the sheep liver enzyme [11] and for that has been excluded [15] and no metal ions are present,
from Candida utilis [12]. The oxidative decarboxy- a feature which distinguishes the reaction from those
lation reaction has been described as asymmetric se- of isocitrate dehydrogenase (IDH) and malic enzyme.
quential for the sheep enzyme with ordered product
release: carbon dioxide first and NADPH last. The pre- Experiments with the sheep enzyme failed to demon-
ferred binding order for NADP+ and 6PG depends strate activity with NAD+ and indicated that it did not
upon the buffer system used; for the sheep enzyme in bind (M Silverberg, personal communication); NAD+

*Corresponding author.

( Current Biology Ltd ISSN 0969-2126 651


652 Structure 1994, Vol 2 No 7

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 three-dimensional structure of the sheep liver en-


zyme has been refined at 2.5A resolution [22] and sub-
sequently with data extending to 2.0A (C Phillips, S Results
Gover and MJ Adams, unpublished data). The enzyme Binary complex structures and refinement
crystallizes in the space group C222 1 ; the dimer has Fig. 2 shows the initial difference maps, contoured at
crystallographic two-fold symmetry. The monomer is il- 3o and 60, calculated using terms (Fcomplex - Fnative)
lustrated in Fig. 1 for reference. Each subunit has three exp ioca, where the phase set for the refined native
domains: the amino-terminal 13-a-3 domain (residues structure, including solvent, was used. There is no ev-
1-176) has a typical dinucleotide-binding fold [23] fol- idence in any difference map of changes in protein
lowed by a short helix and an additional P-ac-03 unit conformation distant from the coenzyme or substrate
antiparallel to that fold; the second domain (residues binding site.
177-434) is the largest, and is helical; the carboxy-ter- In the 2'AMP complex, there is difference density which
minal tail (residues 435-482) burrows through the sec- corresponds to the complete small molecule; in the
ond subunit. Beyond Gly473, the tail is disordered. A two dinucleotide complexes, the adenine, adenine ri-
tightly-bound sulphate ion was identified in the apo- bose, 2'-phosphate and bis-phosphate are seen. Den-
enzyme crystals, which were grown from ammonium sity corresponding to the side chain of Arg33 is also
sulphate at pH6.5. This sulphate is important in mak- visible in all coenzyme difference maps; this side chain
ing inter-subunit contacts and is bound by strictly con- is disordered in the native protein. There is no differ-
served residues in the helical domain of one subunit ence density above 3 for the nicotinamide ribose or
and in the tail of the two-fold-related subunit. Here the nicotinamide in any dinucleotide difference map.
we show that crystals grown in the presence of 6PG The main features of the 6PG difference map are a peak
give a binary complex isomorphous to the apo-enzyme in the internal solvent cavity which is smaller than a
and confirm the suggestion that the 6-phosphate of complete 6PG molecule and a hole corresponding to
6PG displaces the sulphate ion. Two further, less tightly the position of sulphate 507 in the apo-enzyme. The
bound, sulphate ions have been recognized in the apo- peak approaches the position occupied by sulphate
enzyme 2.OA electron density map; one is at a crystal 505 in the apo-enzyme; there is no difference density
contact. The remaining sulphate, in the same pocket as at this position. One additional small peak was visible
the first and approximately 6 A from it, is also displaced in this map which indicated a change in orientation of
by 6PG; it makes three contacts to conserved residues the amide of AsnlO02.
6PGDH coenzyme specificity and mechanism Adams et al. 653

Fig. 1. Monomer of 6PGDH (drawn us-


ing MOLSCRIPT [52]) with domains and
secondary structural elements labelled;
[-strands A-H (blue), ot-helices a-s (red).

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

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 dis-
placed by 6PG are shown. In this figure,
both positive (white) and negative (red)
contours are drawn. All maps used apo-
enzyme phase sets including active site
solvent; they are contoured at 3a and
6a. Standard deviations () were calcu-
lated using the standard errors of the in-
put data [53].

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

Table 1. Refinement statistics.

Complex Resolution Final Parameters of Other comments


limits residual solvent maska

Nbr8ADP+ 20-2.3 A 20.4 % a = 53.37 %


B = 90A2
-3
r = 0.390eA
NADPH 20-2.5 A 17.0% a = 53.51% 24 residues, sulphate
(2.5 A) B = 90 A2 505 and 2 waters
3
r = -0.385 e A- constrained and
omitted/61 residues
and 21 waters
restrained in
intermediate cycles
2'AMP 20-3.17 A 16.9 % a = 53.71% No temperature
B = 95 A2 factor refinement
- 3
r = 0.370e A
6PG 20-2.5 A 17.0 % a = 53.83 % 20 residues and 1
2
B = 95 A water constrained and
3
r = 0.385eA- 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 pro-


tein 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 apo-
enzyme.
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 nu-
cleotides 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.

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 com-
Fig. 3. Final omit/2Fo-Fc maps for (a)Nbr 8ADP + , (b) NADPH 2.5 A plexes, forms the other side of the pocket, shielding
data and (c) 6PG data. The coenzyme or substrate and solvent the adenine from solvent. The plane of the guanidinium
molecules in the binding sites were excluded from the calcula- group is approximately parallel to that of the adenine
tion of Fc. The coenzyme is in the same orientation as in Fig. 2. ring. There is a small additional movement of Phe83
Final refined coordinates are superimposed with coenzyme (and
pyrophosphate) and substrate in atom colours and protein in red. in the Nbr8 ADP + complex to accommodate the larger
These maps are contoured at 1.5cr and 3. brominated adenine. The bromine extends into solvent
656 Structure 1994, Vol 2 No 7

Table 2. Model parameters.

Complex NbrADP+ NADPH 2'AMP 6PG

Standard deviation from ideal:


Bond lengths 0.011 A 0.011 A 0.043 A 0.011 A

Bond angles 2.66 2.53 2.75' 2.37

Dihedral angles 23.0 23.0 22.9 21.9

Improper angles 1.87' 1.35 1.73 1.30
Mean B factor:
2
Main chain 32.6 A 35.1 A2 (27.5 A2) a 30.1A2
2
Side chain 33.9 A2 37.6 A2 (31.3 A2) 32.2 A
Waters included:
Full: number, mean B 320 43.5A2 148 45.9A2 421 62.9A2b 318 48.3A2
Half: number, mean B 100 36.3A2 271 48.7A2 80 38.5A2
Number (%) residues with [,,
in Ramachandran plot:
Most favoured regions 351 (91.6 %) 358 (93.5 %) 348 (90.8 O/o) 354 (92.9 %)
Disallowed regions [481 2 (0.5 /%) 2 (0.5 %) 1 (0.3 %) 2 (0.5 %/o)
Mean coordinate error
(from Luzzati plot [261) 0.30-0.35 A 0.25-0.30 A 0.25-0.30 A 0.20-0.30 A

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 confor-


mation of 6PG in the structure of the
tri-sodium salt (red) and as bound to
6PGDH (blue). Atom types are indicated
by size: C <O <P.

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

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

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

Fig. 6. Stereo pairs showing the bind-


ing 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 conforma-
tions 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 bal-
anced 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. 659

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 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

Table 4. Hydrogen bonds to coenzyme.a Table 5. Hydrogen bonds to substrate.

Coenzyme NbrSADP+ NADPH 2'AMP 6PG region 6PG complex Inorganic ion Apo-enzyme
moiety

1-carboxy O10-0y 5er128 50 4 507 01 Nr Lys183'


Adenine 03-NH Leu10 03-NH Leu10O (09, 010) 01-0 Wat613
ribose 03-N62 Asn32 03-O1 Asn32 03-N62 Asn32' 01-0 Wat614
04-NH Lys75S 04-NH Lys75 04 NH Lys75 09-OC1 Glu190' 02-N62 Asn102*
02 N82 Asn187*
2'-phosphate 02(R)-N82 Asn32* 02(R)-Nrl Arg33b 03-0 Wat886b
01(P)-NE Arg33 04-0 Wat699
01(P)-NT2 Arg33 01(P)-N12 Arg33
02(P)-061 Asn32' 02(P)N62 Asn32' C2-C6a 08-0 Watl109
02(P)-Nc Arg33 02(P-NH Thr34 02(P-Nll Arg33 08-0 Wat1232
02(P-Oyl Thr34 02(P)-Oyl Thr34 02(P) Oy1 Thr34 07 Nc Lys183'
*
03(P)-O1 Asn32* 03(P-061 Asn32 03(P)-01 Asn32* 07-N82 Asn187*
' *b C
03(P)-N82 Asn32 b 03(P)-N82 Asn32 05- N2 His452'#

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

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 Position in fingerprint P1 P2 P3 P4 P5 P6
ctb; it accepts a hydrogen from the main chain nitrogen Consensus sequence NAD Gly X Gly X X Gly
of LeulO in a hydrogen bond and donates a hydrogen Consensus sequence NADP Gly X Gly X X Ala
bond to the main chain carbonyl of Thr34. On binding Sheep sequence 6PGDH Gly Leu Ala Val Met Gly
dinucleotide, the asparagine side chain twists slightly, Residue number 9 10 11 12 13 14
and it is possible for the amide oxygen to accept a Secondary structure PA - - turn ,--- aa -
hydrogen from the 3'-hydroxyl of the adenine ribose
and the amide -NH 2 to donate a hydrogen to a 2'-phos- Fig. 9. NAD(P) fingerprint. Residues in the sheep enzyme that cor-
respond to the fingerprint are shown in bold.
phate oxygen. The main chain nitrogen of LeulO is also
close enough to the 3'-hydroxyl to donate a hydrogen (LDH), alcohol dehydrogenase (ADH) and GAPDH] at
in a hydrogen bond interaction. The hydrogen bonds the end of the strand B. Direct recognition, seen in
seen for the asparagine side chain in the apo-enzyme glutamate dehydrogenase (GDH), involves a hydrogen
are also retained in the binary complexes. bond from the main chain nitrogen of P2 to the 3'-
hydroxyl of the adenine ribose; for indirect recognition,
The small degree of hydrogen bond interaction of the
a hydrogen bond from the main chain nitrogen of P2
bis-phosphate, direct or indirect, is surprising. In both
is made to the carboxyl of the conserved aspartate and
dinucleotide complexes, the negative charge is stabi-
this carboxyl hydrogen bonds both 02' and 03'. In
lized by the dipole of the first helix of the dinucleotide-
6PGDH, Asn32 occupies the position in the fold of the
binding fold (a). The closest oxygen (in the second
conserved aspartate. The hydrogen bond interactions
phosphate) is 4A from the first turn of the helix in
have.features in common with both direct and indirect
the reduced complex and 5.5A away in the oxidized
recognition and the main chain torsion angles are a
complex. The bis-phosphate of NADPH is hydrogen
compromise between the two patterns (Fig. 10). This
bonded through two waters to the main chain nitro-
suggests a range of interactions specifying recognition
gens of the tight A-aa turn and through a third water
between the two extremes; it also suggests that the
to the main chain nitrogen in the 3D-cad turn. The
sixth residue in the fingerprint (glycine in LDH, ADH,
fA-caa turn is a part of the NAD(P) fingerprint [29,33];
GAPDH and 6PGDH; alanine in GDH and in one do-
the sequence for sheep 6PGDH is compared with the
main of GR) is not the only determinant for the recog-
consensus sequences in Fig. 9. The position P3 in the
nition pattern. It is also clear from 6PGDH that an ala-
fingerprint is normally glycine; any side chain of this
nine in position P6 of the fingerprint is not a universal
residue would project into the coenzyme-binding site
discriminator for NADP rather than NAD as proposed
and the C of Alall can be seen in Fig. 6 making
by Scrutton et al. [35].
contact with the bis-phosphate. This interaction, which
renders the amino-terminal turn of ctb less accessible Nicotinamide binding sites; implication for catalysis
to coenzyme, is undoubtedly responsible for the small The conformations of the two dinucleotides bound to
number of hydrogen bond interactions of the bis-phos- 6PGDH involve different interactions with the protein.
phate; it would lower the affinity of the enzyme for The larger number of hydrogen bond contacts made
NAD, thus further enhancing the NADP specificity of by the reduced coenzyme is consistent with its tighter
sheep 6PGDH. binding to the enzyme in solution. The oxidized coen-
zyme makes contacts only with residues in the coen-
Comparison with other NAD(P) enzymes zyme domain, but the nicotinamide of NADPH interacts
Two possible modes of recognition of coenzyme by with conserved residues in cah of the helical domain.
the dinucleotide-binding fold have been described In the substrate complex, these residues interact with
by Baker et al. [34]. They focus on the conserved substrate; in the oxidized coenzyme complex, their in-
glycine-rich turn of the fingerprint and on the aspartate teraction is with the pyrophosphate ion which has re-
[conserved in the sequences of lactate dehydrogenase placed sulphate 507.
662 Structure 1994, Vol 2 No 7

reason is now clear: NADPH displaces the reduced 6PG


I
-

from its binding site by competing for some of its pro-


180- tein ligands. There is already evidence that the reduced
15 coenzyme has a role in decarboxylation or product re-
12 lease [19] and these results give additional weight to
9-1 this evidence.
6 Modelled ternary complex and proposal for oxidation
3 6PGDH is a pro-S (B) dehydrogenase, as is GAPDH
[37]. The conformation of the oxidized coenzyme in
-3 6PGDH resembles that of the NAD + in GAPDH [38]
quite closely, with an rms difference for all atoms
-6
P+
of 1.66A (Fig. 11). The (pro-)chirality of the nicoti-
-90
namides of the bound dinucleotides in the two oxi-
-12 O dation states is different. In solution, pro-S enzymes
-15 have been shown to have the syn conformation. In the
-18 6PGDH-NADPH complex, the coenzyme is positioned
.
-180 '-120 A -60 XA 0
.
60
AA
120
-
180 where it is argued it does not have a redox function
PHI and the ring is anti. In the binary complex with the
oxidized analogue, the ring is however closer to the syn
Fig. 10. Main chain torsion angles for the fingerprint region of sev- than the anti conformation; the si- face of the ring is
eral NAD(P) and FAD enzymes (after [34]). Data were taken from oriented towards the pyrophosphate ion and the sub-
PDB files except for that of glutamate dehydrogenase. A, 1PGD strate binding cleft. The positive charge is stabilized by
(NADP) 6PGDH apo-enzyme [221; B, (NADP) 6PGDH-Nbr 8 ADP+ the pyrophosphate ion. The nicotinamide ring may be
complex (this paper); C, (NADP) 6PGDH-NADPH complex (this pa-
per); D, 1LDM (NAD) lactate dehydrogenase [501; E, 1GD1 (NAD) further stabilized in this position by interaction of its
glyceraldehyde 3-phosphate dehydrogenase [38]; F,1GRA (NADP) it-electron density with the polarizable sulphur of the
glutathione reductase [30]; G, 1GRB (NADP) glutathione reduc- conserved Met13.
tase [30]; H, (NAD) glutamate dehydrogenase [34]; K, 1PHH (FAD)
p-hydroxybenzoate hydroxylase [511; M, 1GRB (FAD) glutathione Superimposition of the structure of bound Nbr 8ADP +
reductase [301. onto the substrate binary complex shows the bound
substrate approaching the si- face of the nicotinamide.
The difference in conformation is first apparent at the Direct hydrogen transfer to NADP + could be achieved,
nicotinamide ribose. The hydrogen bond interaction without protein movement, by adjustments of torsion
made with the reduced coenzyme is not made with angles of the nicotinamide nucleotide portion of the
the oxidized coenzyme and it may be speculated that coenzyme. The Nbr8 ADP + conformation in its binary
this difference is a consequence of the charge differ- complex is compared with a possible active conforma-
ence at the nicotinamide ring. The residues proposed tion, which results from a rotation of 57 about the
as forming hydrogen bonds to the nicotinamide amide C5'-C4' bond of the nicotinamide ribose and smaller
are from different domains in the two complexes. Two rotations about the phosphate bonds, in Fig. 12. The
of the interactions with the reduced coenzyme are with modelled active complex, in which the distance from
residues which bind the substrate in the 6PG binary the 6PG hydrogen to C4 of the nicotinamide is 3 A
complex (Ser128 and Asn187). All residues involved in is shown in Fig. 13.
the nicotinamide interaction in both oxidized and re- Fig. 14 shows the steps of the general base-acid mech-
duced coenzyme complexes are totally conserved. The anism. The 3-hydroxyl of 6PG should approach the
apo-enzyme has been used as a test case for develop- general base when it binds to the enzyme. In the binary
ment of a benzamide probe in the program GRID [36]; complex, the 3-hydroxyl makes hydrogen bonds to N5
the position of the reduced nicotinamide is close to of Lys183 and to N62 of Asnl87: NS of the lysine moves
the energy minimum found, which is rather sharp and by less than 1 A from its position in the apo-enzyme
shows the amide of the probe interacting with His186 and the asparagine 061 by 0.5A; N62 of residue 187
and Glu190 (P Goodford, personal communication). does not move significantly. (The distinction between
His186 is seen to hydrogen bond the nicotinamide N62 and 061 of 187 is apparent in the apo-enzyme
amide in the reduced coenzyme complex; Glu190 is where the nitrogen acts as hydrogen bond donor to
one turn below Asn187 in ah and is less than 4A from an oxygen of sulphate 507.) Thus, N62 of 187 acts as
the nicotinamide. donor in the hydrogen bond to the 3-OH (07) of 6PG;
The reduced coenzyme shows mixed inhibition with the 3-OH must then donate in the hydrogen bond to
respect to the reduced substrate [11]. In the soak- Lys183 N; which can then be seen to be deprotonated.
ing experiment which led to the 2.5A NADPH data In the apo-enzyme and in the Nbr8ADP + complex,
set, we aimed to prepare an abortive ternary complex: Lys183 is protonated and interacts with sulphate 507
enzyme-reduced coenzyme-reduced substrate but a or with the pyrophosphate which replaces it. The pro-
coenzyme binary complex resulted from this soak. The tonation state of Lys183 therefore changes when sub-
6PGDH coenzyme specificity and mechanism Adams et al. 663

Fig. 11. Stereo pair showing the con-


formation of Nbr8ADP + as bound to
6PGDH (red) compared with that of
NAD+ bound to GAPDH (blue). Atom
types are indicated by size:
C <N <O <P <Br.

Fig. 12. Stereo pair showing the con-


formation 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 show-


ing 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 Decarboxylation


for the base in the oxidative stage of the mechanism. The enol-keto tautomerization is the final stage of the
The suggestion that a C-3 alkoxide intermediate may enzyme reaction and it requires a further acidic group
be formed prior to oxidation is consistent with involve- not far distant from C2 of 6PG. Before this stage of
ment of lysine as the base. Decarboxylation is facilitated the reaction, or concomitantly with it if the reaction is
by an acid which will polarize the 3-keto group to yield concerted, the carbon dioxide product will leave the
an ene-diol (or diolate) intermediate. The same lysine binding site. The hydrogen bonds to OF1 of Glu190
residue is able to act as acid in this stage of the reaction. and to Oy of Ser128 will be broken. Although there is
664 Structure 1994, Vol 2 No 7

,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

bonded in such a way that they would take part in any


proton relay in the active site.
Biological implications
Comparison with isocitrate dehydrogenase (IDH) The dehydrogenases of the pentose phosphate
Of the well known NADP-dependent 03-ketoacid de- pathway contribute to its metabolic role by
hydrogenases, the three-dimensional structures are providing reduced nicotinamide adenine dinu-
available for IDH and 6PGDH. Both malic enzyme and cleotide phosphate (NADPH) for synthesis; their
IDH require a divalent metal for activity. There is little NADP specificity is crucial. We have shown three
similarity between the binding site for 6PG in 6PGDH contributions to NADP specificity for sheep 6-
and that for isocitrate in IDH [39] beyond the partici- phosphogluconate dehydrogenase (6PGDH). First,
pation of both subunits of the dimer in both proteins.
Although both enzyme reactions proceed via a general there is extensive interaction of the 2'-phos-
acid-general base mechanism, a magnesium ion stabi- phate with Asn32, Arg33 and Thr34 which follow
lizes an enolate intermediate in the IDH reaction and strand B of the dinucleotide-binding fold. Sec-
the protein base and acid required are shared between ond, ordering Arg33 on binding NADP converts a
subunits; the base has been identified as an aspartate hydrophobic surface depression into an adenine-
(position 283 in the two-fold-related subunit in the E. binding pocket. Third, the alanine which substi-
coli enzyme) and the acid is either a lysine (position tutes for the second glycine of the dinucleotide
230 in the two-fold-related subunit) or a tyrosine (po- binding 'fingerprint' obstructs hydrogen bonding
sition 160). Neither is there similarity in the three-di- between the bisphosphate and the first turn of
mensional structure: IDH does not have a Rossmann helix xta. The binding energy derived from parts
coenzyme fold; the coenzyme and substrate bind in of the coenzyme other than the 2'-phosphate is
a cleft between its two domains which share a mixed
thereby decreased, lowering the affinity for NAD.
sheet.
The arginine interaction is one general method of
IDH is activated by dephosphorylation of a serine in defining NADP specificity.
the substrate-binding site. This serine forms a hydrogen Oxidative decarboxylation of 6-phosphogluconate
bond to one of the carboxyls of isocitrate and the co- (6PG) yields pentose sugars for nucleic acid syn-
valently bound phosphate prevents substrate binding. thesis. 6PGDH is specific for the 6-phosphate;
In contrast, in 6PGDH, phosphate is an inhibitor com- a general base/acid mechanism has been pro-
petitive with substrate, but it is not covalently bound posed in which a protein base deprotonates the
to the enzyme; Ser128 makes a hydrogen bond to the
1-carboxyl of 6PG and is close enough to sulphate 507 6PG 3-hydroxyl. The structure of the co-crystal-
in the apo-enzyme that they form hydrogen bonds to lized 6PG-6PGDH complex defines the role of
the same water (614). The phosphate ions have a sim- conserved residues in catalysis and specificity.
ilar role in the binding sites although there is covalent Residues from the coenzyme and helical domains
modification only in IDH. There is no sequence homol- of one subunit are important in binding and catal-
ogy apparent between the two enzymes. ysis, while close binding to the tail of the second
monomer, which interacts closely with the first,
Summary enhances specificity for the 6-phosphate.
The 2.3A and 2.5A refined structures of the dinu- The coenzyme and substrate binary complexes
cleotide complexes show that there are two ways of
stabilizing the nicotinamide of the coenzyme in this demonstrate that the oxidized coenzyme con-
protein, depending on its redox state. No enzyme con- formation, with a syn nicotinamide, would al-
formational change involving main chain atoms is re- low 6PG binding, whereas the reduced coenzyme
quired or observed for either complex. The bound din- competes with the 1-carboxyl of 6PG. A model
ucleotides share with bound 2'AMP the same protein for direct hydride transfer from the 3-hydrogen
ligands to the adenine ring, 2'-phosphate and adenine to the nicotinamide si- face is proposed in which
ribose. Both coenzyme conformations are extended small movements of the oxidized nicotinamide
and both may represent stages in the enzyme mech- nucleotide have been made which require no pro-
anism: in the oxidized complex, the coenzyme is close tein movement. Lys183 is positioned to act as
to the orientation required for oxidation of the sub- the base facilitating hydride transfer; it is depro-
strate 6PG and in the reduced complex, it is in a po- tonated in presence of substrate but protonated
sition where it may facilitate decarboxylation of the
in apo-enzyme crystals where two sulphate ions
product or CO 2 release. Substrate specificity is achieved
through binding of the phosphate moiety to Tyrl91, bind in the substrate site. Identification of the
Arg287, Lys260 and Arg446 of the second subunit and acid which catalyzes decarboxylation is less cer-
of the carboxyl group to Ser128 and Glu190. Lys183 tain; the most likely candidate is a water hydrogen
and an ordered water molecule are shown to be the bonded to the main chain nitrogen of conserved
most likely catalytic groups. Glyl30. The interactions of NADPH with the pro-
666 Structure 1994, Vol 2 No 7

tein explain its competition with 6-phosphoglu-


Table 6. Conditions of preparation of complex crystals.
conate and are consistent with a possible role in
decarboxylation or product release. Complex Crystals Soak conditions Kinetic parameters

+
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.

Table 7. Data collection parameters.

Complex Sourcea Detector dmax nobsb Residual Completeness F 2 4(crF) mfidd


(wavelength) (nind) Rm()C o/o %0/0

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-
References don.
1. Rosemeyer, MA (1987). The biochemistry of glucose 6-phos- 24. Abdallah, M.A, Adams, MJ., Archibald, I.G., Biellmann, J.-F., Hel-
phate dehydrogenase, 6-phosphogluconate dehydrogenase and liwell, J.H. & Jenkins, S.E. (1979). Binding of coenzyme and
glutathione reductase. Cell Biochem. Funct. 5, 79-95. substrate and coenzyme analogues to 6-phosphogluconate de-
2. Somers, D.O'N., Medd, S., Walker, J.E. & Adams, M.J. (1992). hydrogenase from sheep liver. Eur. J Biochem. 98, 121-130.
Sheep 6-phosphogluconate dehydrogenase: revised protein se- 25. Somers, D.O'N., Hajdu, J. & Adams, MJ. (1991). A two-step pu-
quence based upon the sequences of cDNA clones obtained rification procedure for sheep liver 6-phosphogluconate dehydro-
with the polymerase chain reaction. Biochem J. 288, 1061-1067. genase. Protein Expression & Purification 2, 385-389.
3. Barrett, M.P. & Le Page, R.W.F. (1993). A 6-phosphogluconate 26. Luzzati, V. (1953). Resolution d'une structure crystalline lorsque
dehydrogenase gene from Trypanosoma brucei Mol. Biochem. les positions d'une partie des atomes sont connues: traitement
Parasitol 57, 89-100. statistique. Acta Crystallogr. 6, 142-152.
668 Structure 1994, Vol 2 No 7

27. Tavale, S.S. & Sobell, H.M. (1970). Crystal and molecular struc- 41. Stuart, D.I., Levine, M., Muirhead, H. & Stammers, D.K. (1979).
ture of 8-bromoguanosine and 8-bromoadenosine, two purine Crystal structure of cat muscle pyruvate kinase at a resolution
nucleosides in the syn conformation. J Mol Biol. 48, 109-123. of 2.6A. J Mol Biol. 134, 109-142.
28. Olsen, K.W., Garavito, R.M., Sabesan, M.N. & Rossmann, M.G. 42. Leslie, AG.W. (1992). Recent changes to the MOSFLM package
(1976). Studies on coenzyme binding to glyceraldehyde-3-phos- for processing film and image plate data. In Joint CCP4 and ESF-
phate dehydrogenase. J Mol Biol. 107, 577-584. EACBM Newsletter on Protein Crystallography.26, SERC Labo-
29. Wierenga, R.K., Terpstra, P. & Hol, W.GJ. (1986). Prediction of ratory, Daresbury, Warrington WA4 4AD, UK.
the occurrence of the ADP-binding l3B-fold in proteins, using 43. Briinger, AT. (1992). X-PLOR (version 3.0) Manual. Yale Uni-
an amino acid sequence fingerprint. J Mol BioL 187, 101-107. versity, New Haven, CT.
30. Karplus, P. & Schulz, G. (1989). Substrate binding and catal- 44. Jones, TA (1985). Interactive computer graphics: FRODO. Meth
ysis by glutathione reductase as derived from refined en- ods Enzymol 115, 157-171.
zyme:substrate crystal structures at 2A resolution. J. Mol Biol. 45. Jones, TA, Zou, J.-Y., Cowan, S.W. & Kjeldgaard, M. (1991). Im-
210, 163-180. proved methods for building protein models in electron density
31. Volz, K.W., et al, & Kraut, J. (1982). Crystal structure of avian maps and the location of errors in these models. Acta Crystal-
dihydrofolate reductase containing phenyltriazine and NADPH. J logr. A 47, 110-119.
Biol Chem 257, 2528-2536. 46. Smith, G.D., Fitzgerald, A & Caughlan, C.N. (1974). The crystal
32. Fita, I. & Rossmann, M.G. (1985). The NADPH binding site on and molecular structure of trisodium 6-phospho-D-gluconate di-
beef liver catalase. Proc. Nat Acad Sci. USA 82, 1604-1608. hydrate, Na 3PO 4 .C6H1006 .2H 2O and comparison of results from
33. Hanukoglu, I. &Gutfinger, T. (1989). cDNA sequence of adreno- filtered and monochromatic radiation. Acta Crystallogr. B 30,
doxin reductase; identification of NADP-binding sites in oxidore- 1760-1766.
47. Hodel, A, Kim, S.H. & Bringer, AT. (1992). Model bias
ductases. Eur. J Biochem. 180, 479-484.
in macromolecular crystal structures. Acta Crystallogr. A 48,
34. Baker, PJ., Britton, K.L., Rice, D.W., Rob, A. & Stillman, TJ.
851-858.
(1992). Structural consequences of sequence patterns in the fin-
48. Morris, AL., MacArthur, M.W., Hutchison, E.G. & Thornton, J.M.
gerprint region of the nucleotide binding fold. J Mol. Biol. 228, (1992). Stereochemical quality of protein coordinates. Proteins
662-671. 12, 345-364.
35. Scrutton, N.S., Berry, A. & Perham, RN. (1990). Redesign of the 49. Topham, C.M. & Dalziel, K. (1986). Chemical modification of
coenzyme specificity of a dehydrogenase by protein engineering. sheep liver 6-phosphogluconate dehydrogenase by diethylpyro-
Nature 343, 38-43. carbonate. Evidence for an essential histidine residue. Eur. J
36. Boobbyer, D.NA, Goodford, PJ., McWhinnie, P.M. &Wade, R.C. Biochem. 155, 87-94.
(1989). New hydrogen-bond potentials for use in determining en- 50. Abad-Zapatero, C., Griffith, J.P., Sussman, J.L. & Rossmann, M.G.
ergetically favorable binding sites on molecules of known struc- (1987). Refined crystal structure of dogfish M4 apo-lactate de-
ture. J Med Chem 32, 1083-1094. hydrogenase. J Mol Biol 198, 445-467.
37. You, K.-S. (1985). Stereospecificity for nicotinamide nucleotides 51. Schreuder, HA, Van der Laan, J.M., Hol, W.G.J. & Drenth, J.
in enzymatic and chemical hydride transfer reactions. CRC Crit. (1988). Crystal structure of phydroxybenzoate hydroxylase com-
Rev. Biochem. 17, 313-451. plexed with its reaction product 3,4-dihydroxybenzoate. J. Mol.
38. Skarzynski, T., Moody, P.C.E. & Wonacott, AJ. (1987). Structure BioL 199, 637-648.
of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus 52. Kraulis, P.J. (1991). MOLSCRIPT: a program to produce both
stearothermophilisat 1.8A resolution. J Mol. Biol 193, 171-187. detailed and schematic plots of protein structures. J. Apple. Crys
39. Hurley, J.H., Dean, AM., Koshland, D.E. Jr. & Stroud, R.M. (1991). tallogr. 24, 946-950.
Catalytic mechanism of NADP +-dependent isocitrate dehydroge- 53. Blundell, T.L. & Johnson, L.N. (1976). Protein Crystallography.
nase: implications from the structures of magnesium-isocitrate Academic Press, New York.
and NADP+ complexes. Biochemistry 30, 8671-8676.
40. Howard, AJ. (1988). A Guide to Macromolecular X-ray Reduc-
tion for the Nicolet Area Detection: The Xengen System. Version
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.

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