From the Department of Biochemistry and Molecular Biology,
Wayne State University School of Medicine, Detroit, Michigan 48201 and
the
Interdepartmental Program in Medicinal Chemistry,
College of Pharmacy, University of Michigan,
Ann Arbor, Michigan 48109
Received for publication, August 29, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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3-Deoxy-D-manno-octulosonate-8-phosphate
synthase (KDO8PS) from the hyperthermophilic bacterium
Aquifex aeolicus differs from its Escherichia
coli counterpart in the requirement of a divalent metal for
activity (Duewel, H. S., and Woodard, R. W. (2000)
J. Biol. Chem. 275, 22824-22831). Here we report the
crystal structure of the A. aeolicus enzyme, which was
determined by molecular replacement using E. coli KDO8PS as
a model. The structures of the metal-free and Cd2+ forms of
the enzyme were determined in the uncomplexed state and in complex with
various combinations of phosphoenolpyruvate (PEP), arabinose
5-phosphate (A5P), and erythrose 4-phosphate (E4P). Like the E. coli enzyme, A. aeolicus KDO8PS is a homotetramer containing four distinct active sites at the interface between subunits. The active site cavity is open in the substrate-free enzyme
or when either A5P alone or PEP alone binds, and becomes isolated from
the aqueous phase when both PEP and A5P (or E4P) bind together. In the
presence of metal, the enzyme is asymmetric and appears to alternate
catalysis between the active sites located on one face of the tetramer
and those located on the other face. In the absence of metal, the
asymmetry is lost. Details of the active site that may be important for
catalysis are visible at the high resolution achieved in these
structures. Most notably, the shape of the PEP-binding pocket forces
PEP to assume a distorted geometry at C-2, which might anticipate the
conversion from sp2 to
sp3 hybridization occurring during intermediate
formation and which may modulate PEP reactivity toward A5P. Two water
molecules are located in van der Waals contact with the si
and re sides of C-2PEP, respectively.
Abstraction of a proton from either of these water molecules by a
protein group is expected to elicit a nucleophilic attack of the
resulting hydroxide ion on the nearby C-2PEP, thus
triggering the beginning of the catalytic cycle.
3-Deoxy-D-manno-octulosonate-8-phosphate
synthase (KDO8PS1;
phospho-2-dehydro-3-deoxyoctonate aldolase, EC 4.1.2.16)
catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate (Fig. 1) (1). This reaction is the first
step in the biosynthesis of
3-deoxy-D-manno-octulosonate, an essential
component of the lipopolysaccharide of all Gram-negative bacteria (2).
A reaction very similar to KDO8P synthesis is the formation of
3-deoxy- D-arabino-heptulosonate 7-phosphate
(DAH7P) from erythrose 4-phosphate (E4P) and PEP catalyzed by DAH7P
synthase (DAH7PS; phospho-2-dehydro-3-deoxyheptonate aldolase, EC
4.1.2.15), which constitutes the first step of the shikimate pathway
(3). Earlier studies have established that KDO8PS and DAH7PS share
several mechanistic characteristics. For example, in both enzymes, the
condensation step of the reaction is stereospecific, involving the
addition of the si face of C-3PEP to the
re face of the A5P/E4P carbonyl (4-7). In particular, KDO8PS and DAH7PS are two of only four known PEP-utilizing enzymes in
which phosphate release from PEP occurs by cleavage of the C-O rather
than the P-O bond (UDP-N-acetylglucosamine
enolpyruvyltransferase (MurA) and 5-enolpyruvylshikimate-3-phosphate
synthase represent the other two members of this group). As a
consequence, the anomeric oxygen of the products KDO8P and DAH7P
originates from bulk solvent (5).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Biosynthesis of KDO8P from A5P and
PEP.
The recently determined structures of Escherichia coli KDO8PS and DAH7PS (8-11) provide evidence that these two enzymes are not only mechanistically, but also structurally related, and that they probably originated from a common ancestor. However, although all the known bacterial and eukaryotic DAH7PSs require divalent metals for activity (12, 13), two different groups or classes of KDO8PSs have been recognized with respect to their requirement for metals (14). The most extensively studied member of the KDO8PS family, the E. coli enzyme, does not require a metal. In contrast, KDO8PS from the hyperthermophile Aquifex aeolicus requires a divalent cation for activity, with the level of activation varying over a 10-fold range in the order Cd2+ ~ Mn2+ > Ni2+ ~ Co2+ > Ca2+ > Cu2+ > Mg2+ ~ Zn2+ (15).
To understand the basis for the different metal requirement between
these two enzymes, we have determined the crystal structure of A. aeolicus KDO8PS in the presence of various combinations of
substrates and metal. Results from this study support the previous hypothesis (11) that the reaction of KDO8P synthesis proceeds through
the formation of a linear intermediate and point to the essential role
played by a surface loop in isolating the active site from bulk solvent
and by the geometry of the active site in influencing the electronic
configuration of the substrates during turnover.
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EXPERIMENTAL PROCEDURES |
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Protein Purification and Crystallization-- Recombinant A. aeolicus KDO8PS was isolated from E. coli BL21(DE3) cells harboring plasmid pAakdsA, as described previously (16). A concentrated KDO8PS solution (30 mg/ml in ~15 mM Tris-Cl) was obtained from purified enzyme that had been dialyzed against 5 mM Tris-Cl (pH 7.5), lyophilized, and then reconstituted with distilled water. Lyophilization does not affect the activity of the reconstituted enzyme. Crystals were obtained by vapor diffusion in hanging drops. For this purpose, the concentrated enzyme solution (30 mg/ml) was mixed 1:1 with a reservoir solution containing 100 mM sodium acetate (pH 4.8-4.9) and 5-6% polyethylene glycol 4000. The crystallization trays were incubated at 4 °C for 1-2 weeks. Crystals (typically 0.12-0.15 mm3) were harvested and maintained in a cryoprotectant holding solution consisting of 100 mM sodium acetate (pH 4.8-4.9), 21% polyethylene glycol 4000, 16% glycerol, and 5% ethylene glycol.
The concentrated solution of A. aeolicus KDO8PS as well as crystals of this enzyme in the original crystallization drops have a slight pink color. As discussed by Duewel et al. (15, 16), this color is probably due to the presence of substoichiometric amounts of bound iron in the enzyme produced in E. coli. For these studies, we have opted to collect diffraction data from crystals that were either completely depleted of any metal or in which the iron had been replaced with Cd2+, the ion that confers the highest activity in solution (15). Incubation of A. aeolicus KDO8PS crystals in a cryoprotectant liquor containing 100 µM CdCl2, with several daily changes of this solution, bleaches their color completely within 2 days. We attribute this color change to the replacement of iron by Cd2+. For experiments in which it was necessary to remove any residual traces of metal from the enzyme, crystals were incubated for several days in a cryoprotectant holding solution containing 10 mM EDTA, with frequent changes of the solution. Crystals incubated with EDTA are also completely colorless.
Structure Determination and Refinement--
Crystals of A. aeolicus KDO8PS belong to space group P3121 with unit
cell dimensions a = b 84.3 Å and
c
159.5 Å and diffract to ~1.9 Å. Data sets were
collected at 100 K with an R-axis IV image plate detector or with a
Bruker Hi-Star detector at the CuK
wavelength (see Table
I). Oscillation data were processed with HKL (17) or SAINT (Brucker,
Inc.). The structure of the A. aeolicus enzyme was
determined by molecular replacement using the structure of one monomer
of the tetrameric E. coli enzyme (11) as the starting model.
All the steps of the molecular replacement procedure were carried out
using the CNS Version 1.0 suite of crystallographic programs (18).
Self-rotation and cross-rotation function analyses clearly identified
the presence of two subunits in the asymmetric unit, related by a
2-fold axis of local symmetry. Correct placing of the subunits was
obtained with two consecutive translation searches. Model refinement
was also carried out with CNS Version 1.0 using cross-validated maximum
likelihood as the target function (19). Solvent molecules were added
during the final stages of refinement after the protein model had stabilized.
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RESULTS |
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Overall Structure of A. aeolicus KDO8PS--
The asymmetric unit
of A. aeolicus KDO8PS crystals contains a homodimer. Two
crystal forms of E. coli KDO8PS previously reported by this
laboratory contain instead a homotetramer of the enzyme with 222 local
symmetry (9, 11). A third crystal form reported by Wagner et
al. (10) contains only one chain in the asymmetric unit, but
application of crystal symmetry generates a tetramer essentially
identical to that present in the other two crystal forms. Application
of one of the operations of crystallographic symmetry
(x = y, y = x,
z = z) of space group P3121
produces a tetrameric assembly of A. aeolicus KDO8PS that
can be superimposed on the E. coli enzyme with a root mean
square deviation of only 1.22 Å for 696 aligned C-
atoms. In line
with this finding, comparative analysis of the A. aeolicus
(Mr 29,734) and E. coli
(Mr 30,833) KDO8PSs by analytical size-exclusion
chromatography reveals that the two enzymes have almost identical
elution profiles (16). The enzyme from Salmonella
typhimurium also demonstrates analogous chromatographic behavior
(20). These observations suggest that the native form of KDO8PS might
be a tetramer in all Gram-negative bacteria.
As in the case of E. coli KDO8PS, the A. aeolicus
enzyme adopts a (/
)8-barrel topology (Fig.
2). Each monomer of A. aeolicus KDO8PS differs from the E. coli counterpart
mainly at the N terminus, as the E. coli enzyme has an
additional
-hairpin that seals the N-terminal end of the barrel.
Consequently, the first residue of the thermophilic KDO8PS corresponds
to residue 16 of the E. coli sequence. The enzyme active
sites are located at the C-terminal end of the
-barrel of each
subunit, at the interface with the adjacent subunit. Three long loops,
L2, L7, and L8, control access to the active site cavity (Fig. 2). L7
and L8 were both disordered in the E. coli enzyme (11). L8
is always ordered in A. aeolicus KDO8PS; L7 becomes ordered
under particular conditions (see below).
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Structure of the Substrate-free Enzyme--
The structure of
substrate-free A. aeolicus KDO8PS was determined for the
both the Cd2+ and metal-free forms of the enzyme. The
Cd2+ form (Table I,
Cd2+ column) was intentionally pursued because
Cd2+ is the most effective activator of KDO8PS (15). In the
active site of the enzyme, the Cd2+ ion displays a
distorted octahedral coordination: the thiolate of Cys-11 and the
-nitrogen of His-185 provide two axial ligands, whereas the
carboxylate moieties of Glu-222 and Asp-233 and a water molecule
provide the equatorial coordination (Fig.
3). The water molecule that acts as
equatorial ligand is also stabilized by a hydrogen bond to the
-nitrogen of Lys-46. These features of the active site of A. aeolicus KDO8PS are particularly reminiscent of the homologous
enzyme DAH7PS. The recently reported crystal structure of the E. coli phenylalanine-regulated DAH7PS (8) shows a lead ion
(Pb2+) bound in the active site and interacting with PEP.
The lead ion, which was included in the crystallization for the purpose of determining the structure by multiple wavelength anomalous diffraction, but is itself an extremely poor activator of DAH7PS (8),
is believed to occupy the same active site location as other metals
that are good activators (13). As expected, several residues in the
coordination sphere of the lead ion in DAH7PS (Cys-61, His-268,
Glu-302, and Asp-326) have counterparts in the active site of A. aeolicus KDO8PS (Cys-11, His-185, Glu-222, and Asp-233).
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In the substrate-free Cd2+ form of A. aeolicus KDO8PS, a phosphate ion (herein designated as PO4-1) occupies the position corresponding to the phosphate moiety of PEP in DAH7PS (8) or to the SO4-1 site described in the E. coli enzyme (11) (Fig. 3). A second phosphate ion (herein designated as PO4-2), corresponding to the SO4-2 ion of the E. coli enzyme, is located ~10 Å from PO4-1 in a raised position close to the opening of the barrel and beneath the long loops L2 (residues 44-58) and L7 (residues 187-201) (Figs. 2 and 3). Some broken density associated with L7 is visible in one of the two subunits contained in the asymmetric unit; L7 of the other subunit is completely disordered.
The structure of the substrate- and metal-free enzyme was obtained by
extensive incubation of the crystals in the presence of 10 mM EDTA (Table I, - column). This structure is
essentially identical to that of the Cd2+ enzyme,
suggesting that the presence of the metal does not alter appreciably
the overall conformation of the enzyme. However, small differences are
present in the positions of the side chains of the residues directly
involved in metal coordination. In particular, the water molecule that
was one of the ligands of Cd2+ is no longer visible. L8,
which provides the metal ligand Asp-233, moves slightly away, and a
different rotamer is present in the side chain of Gln-188 (beginning of
L7), such that the carboxylate and amide moieties of these 2 residues
are now in hydrogen bond distance (Fig. 3). One of the largest changes
is observed in the position of the imidazole ring of His-185, which
undergoes a rotation of ~40° around the C--C-
bond. Although
the position of PO4-1 is essentially unchanged,
PO4-2 is not present, possibly as a consequence of the lack
of stabilizing interactions provided by L7. This loop is completely
disordered in both subunits of the metal-free enzyme between Val-187
and Met-200.
Structure of the Enzyme in Complex with PEP--
Crystals of the
metal-free or Cd2+ enzyme were incubated in the presence of
5 mM PEP (Km(PEP) = 40 µM at 60 °C), and their structures were determined
(Table I, PEP and Cd2+/PEP columns, respectively). As
predicted from the analysis of the structure of E. coli
KDO8PS, PEP binds at the bottom of the active site cavity of the
A. aeolicus enzyme, with its phosphate and carboxylate
moieties stabilized by a network of hydrogen bonds and salt bridges
(Fig. 4A). The phosphate group
is anchored by salt bridges to the side chains of Lys-124 and Arg-154
and by a hydrogen bond to the backbone amide of Ala-102. The
carboxylate group forms salt bridges with Lys-41, Lys-46, and Lys-124
and a hydrogen bond with the hydroxyl of Ser-43. Finally, Lys-124 also
forms a hydrogen bond with the bridging oxygen of PEP. PEP binds in a
similar fashion in the active site of the enzyme both in the presence
and absence of a divalent metal. The most notable difference between
the two structures is in the position of the Glu-222 side chain. If
Cd2+ is present, Glu-222 is a metal ligand, whereas in the
absence of the metal, its side chain moves slightly away from the
vacant metal site (data not shown). Interestingly, the water molecule that, in the presence of Cd2+, is one of the metal ligands
is retained even in the absence of Cd2+. This water is
located on the si face of PEP in van der Waals contact with
the substrate C-2 and C-3. The presence of PEP must have a significant
stabilizing role, as this water molecule is not visible in the
structure of the substrate- and metal-free enzyme. A water molecule is
also in van der Waals contact with the re side of PEP (Fig.
4A). The potential role in catalysis of the water molecules
located on both the si and re sides of PEP is
discussed below.
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A striking feature of PEP bound in the active site of KDO8PS is that the geometry of the molecule at C-2 deviates appreciably from the planarity observed in the crystal structures of free PEP in various ionization states (reviewed in Ref. 21): the magnitude of the distortion is provided by the value of the angle between the plane defined by C-2, C-1, and C-3 and the plane defined by C-1, C-3, and O-2, which is ~12° instead of the expected value of 0° (Fig. 4B). This deviation of the PEP molecule from perfect planarity appears to be imposed by the particular geometry of the active site and may be indicative of the fact that the enzyme modifies the electronic configuration of the substrate to favor a nucleophilic attack at C-2PEP (see below).
Structure of the Enzyme in Complex with A5P--
The structure of
A. aeolicus KDO8PS in complex with A5P was determined by
soaking crystals of the Cd2+ enzyme in the presence of 10 mM A5P (Km(A5P) = 8 µM at 60 °C) (Table I, Cd2+/A5P column).
As predicted by Radaev et al. (11), A5P binds with its
phosphate moiety coincident with the position occupied by
PO4-2 in the substrate-free enzyme (see above). The
position of PO4-1 is unchanged with respect to the
substrate-free enzyme. The phosphate moiety of A5P is stabilized by a
salt bridge to Arg-49 and by hydrogen bonds to the backbone and side
chain of Ser-50 (Fig. 5A). In
this structure, L7 is not ordered and therefore does not appear to
contribute significantly to A5P binding. The carbon tail of A5P is
stabilized by a network of hydrogen bonds directed to the substrate
hydroxyls and to the aldehyde moiety, originating from Asp-233, Asn-48,
and PO4-1 and from several water molecules (Fig. 5,
A and B). In particular, C-2-OHA5P
is only 2.9 Å from Cd2+ and can be considered a weak
ligand of the metal. In this fashion, the substrate C-2-OH appears to
replace the water molecule that is a ligand of Cd2+
in other structures of the enzyme. Polar interactions of A5P with the
active site residues are listed in Table
II.
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A5P binds only to one of the two molecules of KDO8PS present in the asymmetric unit. The active site of the other molecule is filled by PO4-1 and PO4-2. Moreover, if crystallographic symmetry is applied to generate the enzyme tetramer, it becomes apparent that the two active sites in which A5P binds are located on the same "face" of the enzyme. This observation is reminiscent of the asymmetry observed in the E. coli enzyme with regard to the binding of phosphate ions at a surface site (11).
Structure of the Enzyme in Complex with PEP plus A5P--
A.
aeolicus KDO8PS has optimal activity at 95 °C and no detectable
activity at 4 °C (16). As crystals of this enzyme are obtained and
maintained at 4 °C, it was deemed possible to bind both substrates
PEP and A5P without turnover. Both metal-free and Cd2+
crystals of A. aeolicus KDO8PS were incubated in the
presence of 5 mM PEP and 10 mM A5P, and the
structure of the enzyme was determined under these conditions (Table I,
PEP/A5P and Cd2+/PEP/A5P columns, respectively). The two
substrates bind in the active site at the same positions they occupy in
the structures of the enzyme in complex with PEP alone or A5P alone,
respectively. However, in the Cd2+ enzyme, A5P binds to
only one of the two active sites contained in the asymmetric unit (as
observed in the structure of the Cd2+ enzyme incubated with
A5P alone), and PO4-2 fills the second active site. In the
metal-free enzyme, A5P binds to both active sites (Fig.
6). In all the cases in which A5P and PEP
bind simultaneously, L7 becomes ordered and isolates the active site
from the external environment. This loop stabilizes the phosphate
moiety of A5P via hydrogen bonds to the backbone and side chain of
Ser-197 (Fig. 6, A and C). Interestingly, the
electron density associated with L7 is better ordered in the metal-free
enzyme than in the Cd2+ enzyme. Likewise, the electron
density of A5P is not well defined in the Cd2+ enzyme, such
that only the positions of the phosphate moiety and the bridging oxygen
can be identified unambiguously (data not shown). This suggests that,
in the Cd2+ enzyme, some residual activity may be present
even at 4 °C, such that a higher degree of motion characterizes the
chemical species possibly involved in catalysis. In the metal-free
enzyme, the density of A5P is reasonably well defined in one of the two
active sites contained in the asymmetric unit and shows that this
substrate presents the re side of its aldehyde moiety to the
si side of C-2PEP (Fig. 6, C and
D), in agreement with previous stereochemical studies
(4-6). In this site, there is no electron density at the position
usually occupied by the water molecule that coordinates Cd2+. In the other active site, the water molecule is
present, but the density of A5P is not continuous between C-2 and C-4
(Fig. 6, A and B). Polar interactions of PEP and
A5P with the active site residues are listed in Table II.
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A |Fo Fc| difference map
of the active sites in the metal-free enzyme shows clearly three small
positive densities surrounding the sulfur of Cys-11. The most plausible interpretation of these densities is that, during the course of the
prolonged incubation of the enzyme first with EDTA to remove any traces
of metal and then with PEP and A5P, the sulfhydryl group (-SH) of
Cys-11 was oxidized to a sulfonic group (-SO3). This
interpretation is supported by other studies carried out with the
soluble enzyme in which it was shown that, in the absence of metal,
Cys-11 becomes prone to
oxidation.2
Structure of the Enzyme in Complex with PEP plus E4P--
It has
been previously reported that E4P, the substrate of DAH7PS, does not
serve as a substrate for A. aeolicus KDO8PS (15, 16).
Furthermore, in the E. coli enzyme, E4P acts as a
competitive inhibitor with respect to A5P.2 Therefore, in
view of the mechanistic and structural similarities between KDO8PS and
DAH7PS, it might be expected that E4P binds in the active site of
KDO8PS in the same position as A5P, but that such binding does not lead
to a condensation product with PEP because E4P is one carbon shorter
than A5P. To verify this hypothesis, crystals of A. aeolicus
Cd2+ KDO8PS were incubated in the presence of 5 mM PEP and 10 mM E4P, and the structure of the
enzyme was determined (Table I, Cd2+/PEP/E4P column). Under
these conditions, PEP binds to both subunits, but E4P binds only to one
subunit. If a tetramer is generated by application of crystal symmetry,
binding of E4P takes place in the two active sites that are visible
from the same side of the enzyme. In these sites, the phosphate moiety
of E4P occupies the same position assumed by the phosphate moiety of
A5P. The electron density for the bridging oxygen and C-4 are well
defined; however, unambiguous assignment of the configuration assumed
by the remaining part of E4P in the active site is not possible. A
plausible interpretation of the electron density is that the aldehyde
carbonyl is hydrogen-bonded to the water molecule coordinating Cd2+ and to one of the hydroxyls of the phosphate moiety of
PEP; C-3-OHE4P and C-2-OHE4P are also in
hydrogen bond distance from the hydroxyl of Ser-197 and from the amide
moiety of Asn-48, respectively (Fig. 7).
As in the case of A5P and PEP, L7 becomes well ordered when E4P and PEP
are both bound and isolates the active site from the external environment.
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DISCUSSION |
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KDO8PS and DAH7PS catalyze similar condensation reactions between
PEP and A5P and between PEP and E4P, respectively. Two mechanisms have
been proposed to explain these reactions. According to the first
hypothesis (22) a water molecule attacks at C-2PEP, whereas
C-3PEP is added to the aldehyde of A5P or E4P; this process
would yield a linear intermediate (Fig.
8, MECHANISM I). According to
the second hypothesis (5, 23), condensation of C-3PEP with
the carbonyl carbon of A5P or E4P is concurrent with an attack by
C-3-OH of the monosaccharide on C-2PEP; this process would
lead to the formation of a cyclic intermediate (Fig. 8, MECHANISM
II). We speculated that two sulfate ions bound in the active site
of E. coli KDO8PS occupy the positions of the phosphate
moieties of PEP and A5P and posited that the distance of 13 Å between
these positions is consistent with a mechanism that proceeds through
the formation of a linear intermediate (11). However, direct
experimental confirmation of this hypothesis was not possible since
attempts to visualize the substrates PEP and A5P by incubating crystals
of E. coli KDO8PS in the presence of these compounds were
hampered by the high ionic strength of the holding solution, which
prevented binding of either substrate. The crystal structure of DAH7PS,
although providing the location for PEP in the active site, did not
contain E4P, hence also could not provide direct experimental
confirmation of our hypothesis.
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In contrast, crystals of the A. aeolicus enzyme are stable in a holding solution of low ionic strength, which allowed the determination of the structure of the enzyme both in its substrate-free form and in the presence of bound PEP and A5P (or E4P). The distance between the phosphate moieties of PEP and A5P (or E4P) in A. aeolicus KDO8PS is only 10 Å, which is the same distance observed in the structure of the E. coli DAH7PS (8) between the phosphate moiety of PEP and a lone sulfate (or phosphate) ion believed to occupy the position of the phosphate group of E4P. A distance of 10 Å between two sulfate ions bound in the active site has been reported also by Wagner et al. (10) for a crystal form of E. coli KDO8PS in which only one chain is present in the asymmetric unit. We had originally postulated that the distance of 13 Å between the two sulfate ions bound in the active site of E. coli KDO8PS (versus 10 Å in DAH7PS) reflected the fact that A5P, the substrate of KDO8PS, is one carbon longer than E4P, the substrate of DAH7PS. This element of our hypothesis is clearly not supported by the observation that, in A. aeolicus KDO8PS, both A5P and E4P are accommodated in the active site without a change in the distance between the phosphate moieties of these substrates and that of PEP. The experimentally observed positions of PEP and A5P (Fig. 6) or of PEP and E4P (Fig. 7) are, however, clearly consistent with the formation of a linear reaction intermediate because the distance between C-2PEP and C-3-OHA5P or between C-2PEP and C-2-OHE4P, is >5 Å. If the reaction took place via formation of a cyclic intermediate, these two atoms would have to be much closer than 5 Å for a bond to be formed between them, without invoking large conformational changes in the active site.
A. aeolicus KDO8PS, like all known DAH7PSs, requires a
divalent cation for activity. Moreover, the coordination of the metal in the A. aeolicus enzyme is very similar to that observed
for the metal present in the structure of E. coli DAH7PS.
What is the function of the metal in these enzymes? One possibility is that it serves a structural function such as that of correctly orienting PEP, A5P (or E4P), or the reaction intermediate in the active
site. With regard to this point, we have not observed significant changes in the position of PEP in A. aeolicus KDO8PS in the
absence or presence of metal. However, changes in the position of PEP might occur during catalysis. For example, in the crystal structure of
the E. coli DAH7PS in complex with Mn2+ and the
PEP analog 2-phosphoglycolate, the octahedral coordination of the metal
is completed by the carboxylate moiety of 2-phosphoglycolate, which
substitutes for PEP in the active site (24). The binding mode of
2-phosphoglycolate might reflect the fact that a direct coordination of
PEP with the active site metal occurs during turnover. Likewise, a
direct interaction between A5P and Cd2+ might occur at some
stage during catalysis. An example of this kind of interaction was
observed in the structure of the Cd2+ enzyme in complex
with A5P (Fig. 5). Thus, two different conformations of A5P have been
observed in the various complexed states of A. aeolicus
KDO8PS. In one conformation, a water molecules coordinates Cd2+ and is also in hydrogen bond distance from both
C-2-OHA5P and C-3-OHA5P (Fig. 6). In the
second conformation, C-2-OHA5P replaces water as a ligand
of Cd2+ (Fig. 5). Thus, although the active site holds PEP
very tightly, it allows significant conformational flexibility to A5P.
It is unlikely that such an active site would have been retained during evolution without a specific reason. An interesting possibility is that
the two conformations of A5P reflect the affinity of the enzyme for A5P
(or for the chemical groups originating from A5P) at different stages
of catalysis. At the start of the reaction, water may be necessary as a
simultaneous ligand of the metal and A5P to favor the formation of a
bond between PEP and A5P (see below). Once the reaction intermediate is
formed, then that part of the molecule that originated from A5P could
be stabilized via a direct interaction with the metal. The requirement
for water at the beginning of the reaction may be rationalized by
noticing that the postulated mechanism of formation of a linear
intermediate predicts that a water molecule attacks C-2PEP
(Fig. 8, MECHANISM I) and that this attack would be favored
by abstraction of a proton from this water by a base. The water
molecule that acts as one of the Cd2+ ligands in A. aeolicus KDO8PS is in van der Waals contact with the si
face of C-2PEP. A water molecule is also one of the
equatorial ligands of the metal in the structure of DAH7PS in complex
with Mn2+ and 2-phosphoglycolate (24). Coordination of
water to a divalent cation is expected to lower its
pKa and to favor its deprotonation to a hydroxide
ion. However, if the function of the metal in A. aeolicus
KDO8PS (and in DAH7PS) is that of activating a catalytic water, how can
E. coli KDO8PS catalyze the same reaction without metal? In
this context, it is probably important that, in the E. coli
enzyme, a water molecule is present in almost the same position as the
water coordinating Cd2+ in the A. aeolicus
enzyme. In the E. coli enzyme, this water is stabilized by
hydrogen bonds to two bases, the -nitrogen of Lys-60 and the
-nitrogen of His-202, that might favor its deprotonation.
If the water molecule that coordinates Cd2+ is involved in
attacking the si face of C-2PEP, then the
formation of the linear intermediate would be the result of a
syn addition of water to C-2PEP and of
C-1A5P to C-3PEP. However, a syn
addition to PEP is unprecedented. Therefore, it is worthwhile to
examine the alternative possibility of a water attack on the
re side of C-2PEP. Such an event might indeed
occur in A. aeolicus KDO8PS because a water molecule is also
present on the re side of PEP in van der Waals contact with
C-2PEP (Figs. 4, 6, and 7). This water is stabilized by a
network of hydrogen bonds that includes one of the hydroxyls of the
phosphate moiety of PEP, Asp-81, and His-83 (Fig.
9). A chain of hydrogen bonds could be
involved in transferring a proton from this water molecule via Asp-81
to His-83, which is a more likely final acceptor than the aspartic
acid. Asp-81 and His-83 are conserved in all known KDO8PSs, and a
glutamic acid is present at the equivalent position of Asp-81 in
DAH7PSs (see multiple alignment of Radaev et al. (11)).
However, a residue equivalent to His-81 that could act as a final
accepting base is not present in DAH7PSs. On the other hand, the
electronic environment of the active site in DAH7PS might raise the
pKa of the glutamic acid carboxylate sufficiently
for it to act as a base. Therefore, as water molecules potentially
capable of attacking C-2 are located on both the si and
re sides of PEP, a direct discrimination between a
syn and an anti attack on PEP is not possible at
this time.
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An additional observation allowed by the high resolution of the structures reported in this study deserves special attention. In all the cases in which PEP is bound in the active site of A. aeolicus KDO8PS, it appears to be distorted to optimally fit in the binding pocket. In particular, the geometry of C-2PEP is intermediate between trigonal and tetrahedral (Fig. 4B). This distortion might be preparing C-2PEP for the conversion from sp2 to sp3 hybridization that will take place during the formation of the linear intermediate. Although the direction of the distortion is such that C-2 bulges out of the si face (Fig. 4B), it is difficult to predict on theoretical grounds whether this would favor a water attack from the si side or from the re side. An additional effect of the distortion is that the dihedral angle between the C-2=C-3 double bond and the C=O double bond of the carboxylate moiety of PEP is different from 0°. It has been theorized that the relative orientations of the two planes in which the C=C and C=O double bonds reside control the reactivity of C-3PEP toward C-1A5P (25), and ab initio calculations indicate that such reactivity should be maximal when the angle between the two planes is 90°. However, nothing is known yet about how solvation effects and the active site microenvironment might change the intrinsic properties and reactivity of PEP as derived from quantum chemistry calculations. Undoubtedly, additional theoretical and experimental work will be necessary to clarify these points.
One of the key observations resulting from our study is that binding of
substrates controls the opening or closing of the active site to the
surrounding environment. When both A5P (or E4P) and PEP are bound
simultaneously, L7 is well ordered and isolates the active site from
bulk solvent. If A5P alone or PEP alone binds, the loop is not ordered,
suggesting that it might be alternating between an open and a closed
conformation. Thus, A. aeolicus KDO8PS is structured
in such a way that its active site remains open until both substrates
are bound sequentially. This is particularly important in view of the
fact that the active site is shaped like a funnel, with PEP bound at
the bottom and A5P bound on top of PEP in proximity of the active site
opening. This observation is consistent with earlier kinetic studies
indicating that the reaction of KDO8P synthesis is an ordered process
in which the binding of PEP precedes the binding of A5P (26). It is
also important that the active site becomes isolated from bulk solvent
during catalysis. This may be needed to create a microenvironment in
which the pKa values of catalytic waters and
specific amino acid groups are different from the values normally
expected in bulk phase equilibria. In view of all of these mechanistic requirements, there must exist a way by which information on the occupancy of the two substrates in the active site controls the conformation of L7. In this context, it may be of relevance that His-185, located at the very beginning of L7, is one of the
Cd2+ ligands. The -nitrogen of His-185 is between 3.3 and 3.6 Å from C-2-OHA5P (in the two conformations
observed for this substrate) and is also in hydrogen bond distance of
the water molecule sitting on the si side of PEP. Also at
the beginning of L7, on the carboxyl side of His-185, Gln-188 is
hydrogen-bonded to C-4-OHA5P (in the conformation of A5P
observed in the metal-free enzyme with PEP also bound). Thus, it is
possible that His-185 and Gln-188 sense disturbances in the delicate
network of hydrogen bonds and hydrophobic interactions surrounding the
substrates and act as transducers of information from the active site
to L7.
Of particular mechanistic interest is the observation that, in the
Cd2+ enzyme, A5P (or E4P) binds only to one of the two
active sites of the dimer contained in the asymmetric unit of the
crystal. Application of crystal symmetry to generate the tetramer
reveals that binding of A5P occurs at the active sites located on only one of the two faces of the enzyme. When A5P binds simultaneously with
PEP, the associated ordering of L7 also takes place only on one face of
the enzyme (Fig. 10). A similar
asymmetry was also observed in the binding of sulfate ions to the
surface of the E. coli enzyme (11, 24). As structures of
KDO8PS in complex with A5P (or E4P) were obtained by soaking crystals
in the presence of this substrate, one would expect an equal binding to
the two faces of the enzyme, if the faces were equivalent. The
observation that only one face binds A5P (although both are equally
exposed to solvent) strongly suggests that the asymmetry is preexistent to the addition of the substrates and that turnover cannot take place
in both faces simultaneously. Thus, at any point in time, the entire
population of the enzyme is committed to bind A5P only on one of its
faces: this situation translates into a structural asymmetry that is
always present in the crystal and, we would argue, also in the soluble
enzyme, but becomes clearly manifest only upon the addition of A5P. We
believe that this hypothesis is consistent with the recent
determination of pre-steady-state burst of phosphate release in the
reaction of KDO8P synthesis catalyzed by the E. coli enzyme,
which indicated that only ~50% of the active sites contribute to the
burst (27). This observation was originally interpreted as an
indication of partial inactivation of the enzyme during the course of
the study. A key role in establishing the enzyme asymmetry is played by
the metal. If Cd2+ is present, a change in conformation
appears to originate in the active sites located on one face of the
enzyme, which prevents binding of A5P in the active sites located on
the opposite face (see structure of the Cd2+ enzyme in
complex with PEP and A5P or E4P). However, in the absence of metal,
information on the occupancy of the active sites of one face is no
longer transferred to the other face. The consequence of this lack of
communication is that the active sites on both faces of the enzyme act
independently and bind A5P simultaneously (see structure of the
metal-free enzyme in complex with both PEP and A5P). Thus, altogether,
the crystallographic studies of KDO8PS point to the existence of a
mechanism of alternating site (or face) catalysis that has been
overlooked by previous kinetic studies.
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ACKNOWLEDGEMENT |
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We thank Dr. S. Ackerman for critical evaluation of the manuscript.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grants AI42868 (to D. L. G.) and GM53069 (to R. W. W.) and by a donation in memory of Michael Cooperman.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1FWN, 1FWS, 1FWT, 1FWW, 1FXP, 1FXQ, 1FX6, 1FY6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ These authors contributed equally to the work.
¶ Recipient of a Natural Sciences and Engineering Research Council (Canada) post-doctoral fellowship.
** To whom correspondence should be addressed. Tel.: 313-993-4238; Fax: 313-577-2765; E-mail: mimo@david.med.wayne.edu.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M007884200
2 Ronald W. Woodard, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: KDO8PS, 3-deoxy-D-manno-octulosonate-8-phosphate synthase; KDO8P, 3-deoxy-D-manno-octulosonate 8-phosphate; PEP, phosphoenolpyruvate; A5P, arabinose 5-phosphate; E4P, erythrose 4-phosphate; DAH7P, 3-deoxy-D-arabino-heptulosonate 7-phosphate; DAH7PS, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; L, loop.
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