From the
Centre for Crystallographic Studies,
University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
and the
Department of Biological Chemistry,
University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen,
Denmark
Received for publication, April 10, 2003
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ABSTRACT |
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INTRODUCTION |
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Based on presently known sequences, DHODs can be divided into two main classes (7). Class 1 enzymes, which are subdivided into class 1A and 1B enzymes, are cytosolic proteins, whereas class 2 enzymes are membrane-associated. The bacterium Lactococcus lactis contains genes that encode DHODs representing subclass 1A and 1B, DHODA, and DHODB. Crystal structures have been determined for both of these without and in the presence of the product orotate (810). DHODA is a dimer formed by two identical PyrD subunits each containing an FMN group (11). The natural electron acceptor for DHODA is fumarate (12), but all DHODs can to some extent use a variety of other electron acceptors such as soluble quinones, dyes, and molecular oxygen (13, 14). For DHODA it has been suggested that the substrate and the natural electron acceptor use the same binding site. Kinetic investigations supported a one-site ping-pong mechanism and showed the second half-reaction to be the rate-limiting step for DHODA (13).
The class 1B enzyme is a heterotetramer consisting of two PyrDB subunits, homologous to the PyrDA subunits of DHODA, and two PyrK subunits (15). The PyrK subunits each contain FAD and an [2Fe-2S] cluster, and the presence of these subunits enables the tetrameric enzyme to use NAD+ as electron acceptor (10, 15). The class 2 DHODs are monomeric enzymes; there are structures known for the human and Escherichia coli DHOD (DHODC) (16, 17). Relative to the class 1 enzymes, they possess an extended N terminus, which plays a role in the membrane association of the enzyme and provides the binding site for the respiratory quinones that serve as physiological electron acceptors (16, 17).
The earliest studies on DHOD from bovine liver (18) and Crithidia fasciculata (19) together with more recent studies on DHODB from Enterococcus faecalis (20) and on DHODC from E. coli (21) describe the stereospecific oxidation of (S)-dihydroorotate to orotate as mediated by an active site base. The active site base abstracts the C5-S proton of dihydroorotate (DHO) followed by hydride transfer from the C6 position of DHO to the N5 of FMN. Based on structural comparisons of class 1 and class 2 enzymes we have suggested that the mechanism for the first half-reaction is highly conserved, and two highly conserved lysines are essential for the catalytic function (17). In the class 1 enzymes the active site base is a cysteine residue (7) while in class 2 enzymes it is a serine (14).
In terms of structure DHODA represents the simplest type of DHOD, with a
common binding site for the two substrates. At the same time DHODA exhibits
properties that are common for DHODs in general, which makes the protein an
excellent model system for detailed investigation of the function and
structure of DHODs. The structure of the dimeric DHODA with orotate bound in
the active site is shown in Fig.
1. Each subunit folds into a (/
)8-barrel
with the prosthetic FMN group situated at the C-terminal end of the
-strands at the top of the barrel and close to the dimer interface. The
reaction product, orotate, stacks on the isoalloxazine ring of FMN in a
favorable position for hydride transfer from the substrate to N5 the
isoalloxazine ring. Cys-130, the active site base, is contained in a flexible
and highly conserved loop (residues 129138), which we refer to as the
active site loop. This loop covers the active site, and it is obvious that it
must undergo movements to let substrate in and product out of the active site
(810).
Cys-130 in the active site loop is only conserved in the class 1 DHODs, but
the loop contains three residues that are totally conserved in both classes,
Ser-129, Pro-131, and Asn-132. The role of the latter residue was assigned
earlier (9), but from our
structural investigations studies we were unable to assign any function to the
two former residues. Another very highly conserved segment of amino acids,
residues 5057 in DHODA, found in the DHOD sequences, forms a fixed loop
delineating the active site in the DHOD structures
(810,
16,
17), which we refer to as the
cis-proline loop. Two of the residues in this loop, Arg-57 and Pro-56
(forming a cis-peptide bond to the non-conserved Leu-55) are
conserved in all DHODs. Their putative functions in catalysis have not been
investigated. In addition to the two loop regions, the alignment of DHOD
sequences and structures revealed a number of other residues conserved between
the DHODs. Two of these (Asn-132 and Lys-43 in DHODA) are important for
catalysis (7). Their side
chains are hydrogen-bonded to the product orotate, and in addition Lys-43
interacts with O4 of FMN (9).
Furthermore, in all the known structures the pyrimidine ring of orotate is
held tightly by hydrogen bonds to three totally conserved asparagines residues
(Asn-67, Asn-127, and Asn-193). Three basic residues, Arg-50, Lys-136, and
Lys-213 are only conserved in class 1A. Their presence close to the entrance
to the active site led us to propose
(9) that they could play a role
in the first step of the reaction by the attraction and proper orientation of
the substrate. Arg-50 is located in the cis-proline loop, Lys-136 in
the active site loop, and Lys-213 at the C-terminal of end of a 310
helix, which we will refer to as the Lys-213 helix. The positions of these
residues in the DHODA dimer are illustrated in
Fig. 1.
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We report here the kinetic characterization of ten mutant enzymes, seven of these represent changes to alanine of residues totally conserved among all DHODs (N67A, N127A, N193A, P56A, R57A, S129A, P131A) and three were constructed to change the surface charge at or near the entrance to the active site (R50E, K136E, K213E). In addition structural investigations at low temperature were made for five of them in the presence or absence of the product orotate (N67A(Oro), P56A(Oro), R57A(Oro), K136E, K136E(Oro), K213E(Oro)). A low temperature structure determination was also performed for the uncomplexed DHODA, as our initial structure determination for the enzyme was a room temperature study and contained Cys-130 in an oxidized form (8). These results from the kinetic and structural investigations have revealed several new characteristics of the enzymatic function of DHODA that have implications for the function of dihydroorotate dehydrogenases in general.
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EXPERIMENTAL PROCEDURES |
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Enzyme concentrations were determined from the flavin content (7) after denaturation of the protein in 1% sodium dodecyl sulfate (SDS) for a few minutes at room temperature. The absorbance measured to calculate the enzyme concentration was based on the extinction coefficient of 12.5 mM1 for free FMN at 445 nm.
Steady State KineticsSteady state kinetic measurements of
the enzyme activity with different electron acceptors were carried out in 0.1
M Tris-HCl, pH 8.0 with 1 mM DHO as substrate under
normal aerobic conditions (Table
I). A Zeiss Specord S10 diode array instrument thermostatted at 25
°C was used to record the spectrum as a function of time. When DCIP served
as electron acceptor the absorbance at 600 nm with an extinction coefficient
= 20 mM1
cm1 was used to estimate the orotate
concentration. With oxygen as electron acceptor the concentration of orotate
was estimated from the absorbance at 278 nm using an extinction coefficient
= 7.7 mM1
cm1, and with fumarate as electron acceptor the
orotate absorbance at 300 nm (
= 3.05
mM1 cm1)
was used to estimate its concentration.
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Determination of Orotate Binding Constants by Red Shift
TitrationsThe marked red shift of the FMN absorbance spectrum
(Fig. 2), accompanying the
binding of orotate to DHODA and quantified by the difference between the
absorbance at 515 and 600 nm
(A515A600), was used to
determine the parameters for binding of orotate to the DHODA enzymes. The
spectrum was recorded before and after each of a series
(13) of successive additions
of orotate to the cuvette and corrected for dilution. The total orotate
concentration ranged from 5 µM to 2 mM, but for the
weak binding enzymes, i.e. the N193A, P56A, and N127A proteins,
concentrations up to 12 mM of orotate were used. To determine the
dissociation constants (KD) for the
enzyme-orotate complexes, the data were fitted to
Equation 1, which assumes a
uniform affinity of the ligand at all binding sites and a binding
stoichiometry of 1:1 between the ligand and the number of binding sites
(21),
![]() | (Eq. 1) |
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However, as illustrated in Fig.
3 the binding behavior did not fully agree with
Equation 1 and in some cases, for
enzymes with KD values below 100 µM
we attempted a fit to Equation 2 (22), which describes the
binding of a ligand (L) to an enzyme with two different types of
binding sites (A and B),
![]() | (Eq. 2) |
![]() | (Eq. 3) |
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Redox PotentialsRedox potentials (Table III) were determined using the xanthine-xanthine oxidase method described by Massey (22) with phenosafranin as reference dye, E0(pH 7.0) = 252 mV. Enzyme concentrations were near 1 mg/ml in 0.05 M sodium phosphate, pH 7.0.
CrystallizationPrior to crystallization the proteins were dialyzed against a 25 mM NaH2PO4 buffer, pH 6.0, containing 10% glycerol. The P56A, R57A, N67A, K136E, and K213E mutant enzymes were crystallized with the reaction product orotate in 5-fold molar excess relative to the protein concentration. The native DHODA and the K136E mutant were crystallized also without orotate; in these cases 1 mm DTT was added to the crystallization mother liquor, to ensure a reduced state of the active site cysteine. Good quality crystals were obtained after microseeding with crystals resulting from the original conditions (11) in drops in which no spontaneous crystallization occurred. Optimal microseeding conditions differed slightly from mutant to mutant, but were all within the range 2335% polyethylene glycol 6000, 0.2 M sodium acetate, 0.1 M Tris-HCl, pH 7.59.0, and protein concentrations of 1320 mg/ml.
Data Collection and ProcessingAll data sets were collected
under cryogenic conditions (120 K). Mother liquor containing 10% glycerol was
used as cryoprotectant. The data sets were integrated and scaled using DENZO
and SCALEPACK (23). Structure
factors were derived from the reflection intensities using TRUNCATE
(24). The main statistics for
data collection and integration are given in
Table IV. The crystals of the
mutated proteins belong to the same space group (P21) as the native
enzyme. The unit cell parameters are similar and slightly smaller than those
obtained earlier for the structures at room temperature
(8,
9). The average of the cell
parameters were a = 53.04 ± 0.22 Å, b = 108.36
± 0.24 Å, c = 66.01 ± 0.26 Å and =
103.8 ± 0.11°.
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RefinementAMoRe (25) was used to place the existing model of the native enzyme (8) in the unit cell. To generate the P56A(Oro) and N67A(Oro) structures the refinement was then carried out using REF-MAC (26) and ARP (27) applying tight restraints between the two NCS-related molecules of the dimer. For all the other structures the refinement was carried out with CNS (28) using the automatic procedure for water insertion and restrained NCS. In two structures, K136E and native, NCS was released for parts of the structure, while in R57A(Oro) the NCS restraints were totally released in the final refinement steps. O (29) was used to introduce the mutations and for manual rebuilding. The final refinement statistics are given in Table IV. In all structures more than 90% of the residues are found in the allowed regions of the Ramachandran plot (30), with no residues in the disallowed regions. The illustrations of the molecular structures were prepared using RASMOL (31), MOLSCRIPT (32), and RASTER 3D (33).
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RESULTS |
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Interaction with the SubstrateTable I gives the steady state activities for the wild-type and mutant enzymes determined with three different electron acceptors. When considering these data, it should be kept in mind that the first half-reaction for the wild-type enzyme, i.e. the reduction of the flavin by DHO, is much faster (>50-fold) than the second half-reaction, which is the reoxidation of the flavin by the electron acceptor (7). This means that changes of the amino acid sequence that affect the first half-reaction may not necessarily impair the steady state activity of a mutant enzyme. A few features of the steady state activities are worth noticing. First and as expected, major effects on the activities were produced by alanine mutations of the asparagines (Asn-67, Asn-127, and Asn-193), which form hydrogen bonds to substrate and product in the active site (9). Second, and more surprising, it turned out that a change of the conserved Pro-56 in the cis-proline loop to alanine strongly reduced the steady state activity with all three electron acceptors, while a change of the neighboring and also conserved Arg-57 to alanine increased the steady state activity of DHODA with all three electron acceptors, indicating an important and complex role of the cis-proline loop for optimal function of the active site. Third, it appeared that the changes of the two fully conserved residues (Ser-129 and Pro-131) that flank the active site basic residue (Cys-130) to alanines only moderately impaired the steady state activity of DHODA; while the P131A mutation makes the steady state activity independent of electron acceptor choice and may thus have a strong effect on the rate of the first half-reaction the S129A substitution primarily reduces the activity with fumarate as electron acceptor. The fourth feature of interest in the steady state data in Table I is that of mutations R50E, R136E, and K213E that influence the surface charge of the enzyme, only the K213E mutation interferes significantly with the steady state activity.
Table II shows the KD values for binding of orotate to wild type and DHODA enzymes and the apparent Km and Vmax values for the saturation of the enzymes with DHO and fumarate. Generally, there is a good correlation between the dissociation constants (KD) for the orotate enzyme complex and the Km for DHO as the mutant enzymes that bind orotate poorly generally are difficult to saturate with DHO. However, the mutant enzyme N67A, which displayed an unusual absorption spectrum in the presence of orotate appears to bind orotate almost as strongly as wild-type DHODA, while it has a very high Km for saturation with DHO. It is also remarkable that a change of the residue Lys-136, which we originally expected to be involved in attracting DHO to the active site, produces an enzyme that binds orotate more strongly than the wild-type enzyme does.
The R57A mutant enzyme is unusual since it has increased activity with all electron acceptors, (especially DCIP) and the Km values for saturation with DHO and fumarate are increased 35-fold. It is also remarkable that two mutant enzymes, N193A and N127A, which have lost hydrogen bonds to the substrate (DHO), although stimulated by low concentrations of fumarate (0.25 mM), are inhibited by higher concentrations of fumarate (Tables I and II). This may indicate a competition between the substrate (DHO) and the electron acceptor (fumarate) for binding in the active site of DHODA in line with the observations of Björnberg et al. (13).
In general there is a large variation of the steady state parameters for the different electron acceptors among the mutant enzymes. Fumarate is the natural substrate for DHODA, and it has been supported by kinetic studies that it binds at the site as orotate. DCIP is a much larger molecule and the electron transfer to this molecule cannot be envisioned to take place by direct interaction with the flavin group but rather through more complex electron transfer pathways. Conversely oxygen is a very small molecule and a much poorer substrate that can bind at numerous places at the protein, which opens the possibility for a variety of different electron transfer pathways.
The kinetic data referring to the first half-reaction of some of the DHODA enzymes (including the wild type) are shown in Table III together with the redox potentials of the enzyme-bound flavin. The KD values for saturation of the enzymes with DHO in the first half-reaction varied only by a factor of 3 between the different enzymes, but several of the mutations created significant variations in the maximal bleaching rate (kc) at saturating concentrations of DHO. Most hampered was the K213E mutant enzyme for which kc was reduced about 60-fold, resulting from an unexpected influence of Lys-213 on positioning of the active site flexible loop (see below), and the P131A enzyme for which kc was reduced by about 30-fold, indicating also a crucial role for residues that flank the catalytic Cys-130 in the flexible loop. We find it however, amazing that the mutation S129A only gives a 6-fold reduction of the maximal bleaching rate (kc), since Ser-129 is conserved in all DHODs as well as in the dihydropyrimidine dehydrogenases, enzymes that reduce pyrimidine bases as the first step in pyrimidine breakdown.
The flavin group shows a remarkable constant redox potential in the mutant enzymes close to the 245 mV seen for the wild-type enzyme, the most negative potential being 263 mV displayed by the P131A mutant. Thus changes of redox potential do not explain the observed changes of kinetic parameters of the mutant enzymes.
Crystal Structures
Cryogenic techniques have allowed us obtain a higher resolution structure
of the uncomplexed native enzyme. This structure was solved from crystals
grown in the presence of DTT to avoid oxidation of the active site Cys-130
that was observed previously in the room temperature structure
(8). We wanted to exclude the
possibility that the closed conformation of the active site loop observed in
the uncomplexed native DHODA structure is due to an oxidized active site
cysteine, preventing the loop opening. This new structure showed unambiguously
that no oxidation had taken place and that the active site loop adopts the
same conformation as in the previously reported structure
(8). The higher resolution
enabled us to identify an octahedrally coordinated Mg2+
ion at the C-terminal end of helix 1 in each of the subunits. The
Mg2+ ions were located in electron densities that
previously had been refined as water molecules. The presence of the
Mg2+ ions cannot be related to the function of DHODA,
their role is rather to neutralize the helix dipole.
The structures of the mutant enzymes containing orotate share the common feature that they maintain most of the direct interactions with orotate seen in the native structure (9) with K213E(Oro) as the only exception (see below). The new high resolution structure of the native DHODA is shown in Fig. 1 together with the structures determined for two of the mutant enzymes having orotate bound in the active site K213E(Oro), P56A(Oro), and the structure of the unliganded K136E enzyme. N67A(Oro), R57A(Oro), and K136E(Oro) are not shown as they appear essentially identical to the native in this representation. Though the structures were determined at essentially the same temperature, they show significant variations in their mobility as indicated by the variations in B-factors and conformations of the active site loop. The most remarkable result is displayed by K213E(Oro) structure, which shows the active site loop in an open conformation similar to the one previously seen in the structure of native DHODB (10). The active site loop in the K136E structure was modeled in two different conformations, one corresponding to the closed form of the native DHODA and another to a partially open form. In the following a description will be given of the structures of the mutant enzymes according to their position in the structure and to their order of appearance to the approaching substrate (Fig. 1).
Mutations That Affect the Surface Charges: R50E, K136E, and
K213EThe surface area of DHODA close to the active site contains
three positively charged residues, Arg-50, Lys-136, and Lys-213, which are
conserved among the class 1A DHODs. These residues were genetically changed to
glutamate residues in order to probe the effect of changing charges adjacent
to the entrance of the active site. The stopped flow kinetic experiments
showed that the modified enzymes all exhibit decreased bleaching rates of the
flavin cofactor compared with the native enzyme, and that the effect is most
pronounced for the K213E enzyme (Table
III). This may be taken as a support of our original assumption
that these residues conserved in class 1A play a role in the first step of the
reaction through attraction of the substrate; however, the structure of the
K213E mutant protein provided the unexpected result that the active
site loop is most stable in an open conformation even with orotate bound in
the active site (Figs. 1 and
4a). This dramatic
structural change is associated with alterations in the hydrogen-bonding
system around the active site as illustrated in
Fig. 4b. In the native
structure the ammonium group of Lys-213 is hydrogen-bonded to Asn-197
O1 and to the carbonyl group of Pro-131, which is conserved among all
DHODs. The side chain of Lys-213 is also interacting through water molecules
with the backbone atoms of Phe-216 and Val-133, and with the side chains of
Asn-132 and Ser-68. These interactions seem to maintain a closed active site
loop. The change of the side chain from a hydrogen-bond donor to a
hydrogen-bond acceptor in the K213E mutant enzyme leads to a loss of the
hydrogen bonds that connects the active site loop to the Lys-213 helix in the
native structure, which explains why the active site loop adopts an open
conformation (Fig.
4a).
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At the orotate binding site the hydrogen bond between Asn-132 and orotate is missing in the K213E(Oro) structure, as it cannot be formed with the active site loop in the open conformation. The open loop conformation explains why both dihydroorotate and orotate are poorly bound in the K213E mutant, as shown by the higher dissociation constants in Table II. Furthermore with the active site loop in the open conformation the catalytic Cys-130 is in a position that is far from optimal for catalysis, explaining the strongly reduced bleaching rate of the K213E mutant enzyme in the first half-reaction (Table III).
The side chain of Lys-136 displays a variety of conformations in the different structures, where its only hydrogen-bond partners are water molecules. It is either oriented toward the solvent or points toward Asn-127 in the orotate binding site, which originally gave us reasons to believe that it could be important for substrate guidance and delivery to the active site. A hydrogen bond formed between the Lys (or Glu)-136 backbone NH and the carbonyl group from Val-133 is conserved in all the DHODA structures. In the K136E structure the active site loop in one of the subunits could be refined in two conformations (Fig. 1). One of these (70% populated) corresponds to an intermediate conformation between open and closed, and the other (30% populated) to the closed conformation found in the other subunit. The small decrease in the dissociation constant for orotate and a Km for dihydroorotate equivalent to the native enzyme suggests that the role of Lys-136 is not to attract the substrate as we originally proposed (9). The presence of the extra half-open loop conformation could imply that substitution of a Lys with a shorter and negatively charged side chain may interfere with the mechanism of loop opening and provides an alternative explanation for the decreased bleaching rate. The increase in the orotate affinity suggests that is has become more difficult for orotate to leave the active site in the K136E mutant, but it is hard to say whether this is related to the intermediate conformation of the active site loop in the K136E structure.
Arg-50 is in the same position and makes similar interactions in all the DHODA structures, where it faces the active site loop from the opposite side relative to Lys-213 (Fig. 1). This arginine residue is conserved in DHODB (Arg-55), while in DHODC it is replaced by Gln-92. Close to Gln-92 are both Lys-144 and Arg-90 whose positively charged side chains are located in the same area as Arg-50 in DHODA, suggesting that the presence of a positive charge is important. This is to some degree supported by the kinetics of the R50E mutant. A negative charge in this region may induce repulsion of the substrate and affect formation of the Michaelis-Menten complex, resulting in decreased affinity for substrate and product, whereas the other kinetic parameters are only affected marginally.
Mutations in the cis-Proline Loop: P56A and R57ATwo adjacent residues in the cis-proline loop (Pro-56, Arg-57) are conserved among all DHODs, and the cis-peptide bond connecting Pro-56 to the preceding residue is also fully conserved in all known DHOD structures. Considering that the backbone fold in the P56A(Oro) and R57A(Oro) structures is virtually identical to the structure of the native enzyme, it was surprising that the mutant enzyme P56A has lost most of its activity, whereas the R57A protein appears more active than the native enzyme in particular with DCIP as the electron acceptor (Tables I and II). Orotate binds very poorly to the P56A mutant enzyme, but this is reasonably easily saturated with dihydroorotate (Km is increased by about 3-fold), because the enzyme also has a very low (50-fold decreased) catalytic activity. Replacement of Pro-56 with an alanine residue does not seem to affect the cis-peptide bond or the direct interactions between DHODA and orotate.
The active site loop has a poorly defined electron density in the P56A(Oro) structure, which made it impossible to identify the side chains of residues 133 to 136. The temperature factors, in the active site loop, and in general are particularly high in this structure (Fig. 1). The b and c cell dimensions are significantly longer (1.8 standard deviations) than the calculated average for the low temperature structures, indicating a slightly looser packing of the crystal. Furthermore though the P56A mutant shows the same degradation pattern with trypsin as the native enzyme, it is degraded faster (data not shown). Prolines are known to exert a rigidifying effect on loop structures, so an increase in mobility caused by a substitution of a proline with an alanine is not unusual. It is difficult to explain however how the higher flexibility is transmitted from the cis-proline loop to the active site loop and the rest of the structure, given that the cis-proline loop itself is not particularly disordered in P56A(Oro). The increased mobility in particular of the active site loop, seen in relation to the reduced activity of the P56A mutant could imply that the active site base is not in its optimal position for catalysis. In the native structure the Arg-57 side chain interacts with the carbonyl groups of Glu-51 and the totally conserved Asn-53 via its NH1. The carbonyl group from Arg-57 is hydrogen-bonded to the backbone of Ser-68, which interacts with the side chain of Asn-53. Asn-53 is hydrogen-bonded to the carbonyl group of Asn-132, another totally conserved residue in the active site loop. We originally thought that these interactions could contribute to stabilization of the cis-proline loop and ensure correct orientation of residues in the active site loop. The kinetic results showed on the contrary an increased catalytic activity of the R57A mutant enzyme. There are however no significant differences between the coordinates or thermal mobility of the native and the R57A(Oro) structures so the structural basis for the increased catalytic rate of the R57A mutant enzyme remains elusive.
Mutations Flanking the Catalytic Base Cys-130: S129A and P131ATwo residues, Ser-129 and Pro-131, which flank the catalytic base, Cys-130, were also selected for mutation and changed to alanines. Both of the residues are conserved in all DHODs and in the dihydropyrimidine dehydrogenases, which carry out an inverse reaction, namely the reduction of uracil and thymine during breakdown of pyrimidine bases. Although there is no structural information about these two mutant enzymes (S129A and P131A) information about the structural role of the residues and their significance for catalysis can be inferred from the structural investigations of the other mutants.
The comparison between the K213E(Oro) and the native structure
revealed some important features for the conserved Ser-129. In the native
structures Ser-129 O forms a hydrogen bond to the carbonyl group of
Lys-164, one of the two totally conserved Lys residues that interact with the
FMN group. Furthermore Ser-129 O
is hydrogen-bonded to Asn-193, which
interacts with orotate. With the active site loop in the open conformation
these direct interactions are not possible, but they occur via a water
molecule and Ser-129 O
is instead hydrogen-bonded to Gln-138 O
1.
From the comparison with the DHODB and DHODC structures, we have proposed that
this serine and Gln-138 in DHODA act as hinges of the active site loop
(17). The exchange of the
Ser-129 with alanine leads to the loss of the hydrogen bond formed by the side
chain and must affect the loop movement and orotate binding site.
The S129A mutant enzyme had a decreased binding affinity for orotate and DHO (Tables I and II) and the bleaching rate constant was 6-fold reduced (Table III). The specific activity was almost normal, except when fumarate served as electron acceptor, in which case it was 5-fold reduced.
Like the K213E mutation the P131A mutation generated a decreased affinity for both orotate and DHO and a drop in the reaction velocity (Table I and II). The K213E(Oro) structure shows that the carbonyl oxygen of Pro-131 is important in stabilizing the closed loop conformation. Although the backbone interactions should be maintained in the P131A mutant, prolines tend to stabilize loop structures and the substitution by an alanine residue might increase the flexibility of the active site loop, disrupting the interaction with Lys-213 and favoring the open loop conformation.
The Active Site Mutants N67A, N127A, and N193AThese mutant
enzymes were constructed in order to examine the influence of the aspargine
residues (Asn-67, Asn-127, Asn-193) interacting with the pyrimidine ring of
the product/substrate as revealed from the structure of the DHODA-orotate
complex. The side chains of two of these (Asn-67 and Asn-193) form cyclic
hydrogen-bonded systems with pyrimidine atoms N1, O2, and N3, O4,
respectively, whereas only the amido group of the side chain of Asn-127 is
hydrogen-bonded to O4 of orotate. In accordance with these interactions the
N127A mutant displays the smallest changes of kinetic parameters among the
three mutant enzymes (Tables I
and II). The
Km values for saturation of the N67A and N193A
enzymes are very high and the maximal reaction velocities are somewhat
decreased, but not dramatically. A remarkable feature of the N67A mutant
enzyme is its ability to bind orotate much better than the other two enzymes
with a dissociation constant comparable to the native enzyme
(Table II). The N67A(Oro)
structure provides an explanation for this ability.
Fig. 4c illustrates
how the replacement of Asn-67 with an alanine residue leaves sufficient space
for two water molecules to occupy the space in the active site otherwise taken
by N2 and O
2 of the side chain. The water molecule found close
to the position of N
2 is hydrogen-bonded to orotate O2 and to the
carbonyl group of the substituted Ala-67, the other water molecule fulfills
the same hydrogen bonds as O
2 from Asn-67 in the native structure and
is also hydrogen-bonded to the carboxyl group of orotate and the backbone NH
group of Met-69. These interactions anchor orotate almost as well in the
active site of the N67A mutant as in the native enzyme. The very unusual red
shift in the FMN spectrum of the orotate-bound enzyme
(Fig. 2) can be explained by
the differences in the hydrogen bonds involving water molecules. The spectra
for the N127A and N193A mutant enzymes upon addition of orotate
(Fig. 2) showed a red shift
similar to the one seen for the native enzyme, which suggests that it has not
been possible to replace the side chains of the asparagines residue with water
molecules in these structures. Consistent with the observation that Asn-127
and Asn-193 are contained in a more tightly packed part of the active site
than Asn-67 (9).
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DISCUSSION |
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The role of the conserved residues Pro-56 and Arg-57 in the cis-proline loop is harder to pinpoint precisely. Substitution of these residues conserved in all DHOD classes with alanine affects the catalytic activity, consistent with their high conservation and with the observation from the crystal structures that the active site loop and the cis-proline loop are intimately connected (Fig. 1). The effects on the activities of the two variants was however unexpected. While the activity of the P56A variant is impaired, the R57A variant shows an increase in activity despite reduced affinity for substrate and product. The structures of the two mutant enzymes are very similar to the native enzyme and do not provide a direct structural explanation for the effects of the mutations. Since they deviate in their mobility of the active site loop, which is less mobile in the R57A structure, it is tempting to attribute their enzymatic differences to the difference in mobility. The increased mobility in P56A may influence the position of the catalytic Cys-130, so it is not optimally positioned for catalysis causing the lower activity.
It had been hypothesized that the positively charged residues Arg-50, Lys-136, and Lys-213 have a role in substrate guidance (9). The variant DHODAs where these residues are mutated show a decreased bleaching rate, indicating that the first half of the reaction has been affected. Though this could be interpreted as consistent with the substrate guidance role, the other kinetic and structural results show that this role is only likely for Arg-50. K136E has similar or increased affinity for DHO and orotate compared with native DHODA, which seems inconsistent with a role in substrate guidance. The open active site loop unexpectedly encountered in the K213E(Oro) mutant enzyme suggests an alternative explanation for the decreased bleaching rate, namely an impaired mechanism for controlling loop opening and closing. In the native enzyme the active site loop is closed, and the closed conformation depends on a hydrogen bond interaction of between Lys-213 and the carbonyl group 10 of the conserved Pro-131. Both the highest resolution native DHODA structure and R57A(Oro) contain some residual density too weak to be modeled, which could suggest that a very small fraction of the protein has the active site loop in the open conformation. The closed conformation of the active site loop appears to be the more stable but it is at the same time very mobile. As dihydroorotate approaches the active site, possibly guided by Arg-50, a transient interaction between Arg-50 and Lys-213 might favor temporary opening of the active site loop to let the substrate enter the catalytic site. In K213E the active site loop is most stable in an open conformation, which allows substrate to enter the active site but is not optimal for catalysis. Substitution of Pro-131 with Ala has similar kinetic consequences as substitution of Lys-213. So the open active site loop conformation might also be favored in this case because the hydrogen bond between Lys-213 and the backbone of residue 131 is destabilized by increased loop flexibility.
The active site loop moves around the hinges Ser-129 and Gln-138 in DHODA.
In the closed loop conformation, the backbone NH group of Ser-129 is
hydrogen-bonded to Asn-127 O1, and the Ser-129 O
atom is
hydrogen-bonded to Asn-93 N
2, two residues involved in orotate binding.
With the loop open Gln-138 O
1 is hydrogen-bonded to Ser-129 O
.
The mutation of Ser-129 to alanine affects one of the hinges of the active
site loop by destroying the side chain hydrogen bonds, which could be the
explanation for the low reactivity of the S129A mutant.
Comparison between DHODs from Different ClassesThe structural alignment of representatives from the different classes of DHODs, i.e. DHODA, DHODB, and DHODC (17) showed that Lys-213 only is conserved among the class 1a enzymes and that the open conformation seen in the K213E(Oro) structure corresponds to the conformation of the loop observed in the structure of the uncomplexed class 1B enzyme, DHODB (10). In the DHODB structure in complex with orotate the loop is in a closed conformation, but could only be traced partially, while in the open conformation it could be traced completely, indicating that it moves to carry out the reaction but is more stable in the open conformation. The superposition of DHODA on the DHODB structure shows that Lys-213 in DHODA has no match in DHODB; however, the same space is occupied by Asn-215 and Ile-216. None of these two residues are able to form the same hydrogen bonds as the lysine suggesting that the absence of a lock system in DHODB to be the origin of the difference observed in stability of the loop conformations between DHODA and DHODB. In both DHODA and DHODB the active site loop alternates between an open and closed conformation due to dynamic motion. The loop is locked into the closed conformation in DHODA by Lys-213, but as DHODB lacks this residue its open loop conformation is the more stable. The importance of the closed loop in DHODA is likely to associated with the fact that the substrate DHO and the electron acceptor use the same binding site, meaning that the closed loop conformation is necessary for both half-reactions. In DHODB this is not the case as substrate and electron acceptor binding sites are widely separated and situated in different subunits.
DHODC does also not contain a residue equivalent to Lys-213, and the corresponding protein segment is much shorter. Due to small structural differences in the domains surrounding the active sites of DHODA and DHODC and the presence of the extended N-terminal in DHODC, we would not expect the DHODC active site loop to open in the same way as in DHODB and DHODA (17). The structural differences around the active site loop and the monomeric state of the enzymes suggest that the class 2 DHODs have developed another mechanism for controlling the loop motion.
Subunit AsymmetryAnother result from the kinetic and structural studies is the apparent asymmetry in substrate binding at the two active sites of the DHODA dimer, resulting in an apparent negative cooperativity in ligand binding. The subunits in K136E differ, one subunit has a closed active site loop, while the other shows two conformations of the active site loop. It is noteworthy that the proportions of the two populations are the same as the proportions of low and high affinity sites derived from the kinetic data. These results provide the first structural indications for a non-equivalent behavior of the active sites in the two subunits. We favor the idea that the ligand binding to the first subunit alters the equilibrium between conformations of the active site loop in the second subunit.
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CONCLUSIONS |
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FOOTNOTES |
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* This work was supported by grants from the Danish National Research
Foundation, the Danish National Science Research Council, and the Carlsberg
Foundation. The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
¶ To whom correspondence should be addressed: European Synchrotron Radiation Facility, B.P. 220 Grenoble Cedex, France. Tel.: 45-35-32-0282; Fax: 45-35-32-0299; E-mail: sine{at}ccs.ki.ku.dk and slarsen{at}esrf.fr.
1 The abbreviations used are: DHOD, dihydroorotate dehydrogenase; DHODA,
L. lactis dihydroorotate dehydrogenase A; DHODB, L. lactis
dihydroorotate dehydrogenase B; DHODC, E. coli dihydroorotate
dehydrogenase; DHO, (S)-dihydroorotate; DCIP, 2,6-dichloroindophenol;
DTT, dithiothreitol; NCS, noncrystallographic symmetry.
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ACKNOWLEDGMENTS |
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REFERENCES |
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