From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
Received for publication, March 17, 2003 , and in revised form, May 16, 2003.
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ABSTRACT |
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INTRODUCTION |
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UPPs from Escherichia coli is a dimer of identical subunits of 253
amino acids. The three-dimensional structure of the apoenzyme reveals an
elongated tunnel-shaped active site crevice surrounded by two -helices
and four
-strands (11).
Previous site-directed mutagenesis studies suggested that the substrates FPP
and IPP are bound on top of the tunnel, and the farnesyl moiety of FPP
migrates toward its bottom during product chain elongation
(1416).
The tunnel is sealed at the bottom by the side chain of Leu-137
(11). In the crystal structure
of UPPs from Micrococcus luteus, a sulfate ion bound to a conserved
structural P-loop represents the location of the pyrophosphate moiety of FPP
(10).
The previous E. coli UPPs crystal was grown using polyethylene
glycol (PEG), and no sulfate ion was observed
(11). Metal ion was not found
in both apo-UPPs structures, although the enzyme requires
Mg2+ for activity. Two protein conformers of the E.
coli UPPs were observed, i.e. one with a bound PEG fragment that
adopts a narrower conformation than the other one with water molecules in the
active site, indicating the possible open and close mechanism for substrate
binding and product release. These two conformers have the most striking
difference in the position of the 3 helix, which is connected to a loop
containing amino acids 7282. In the proposed catalytic model, the loop
may play an essential role in pulling the
3 helix toward the active
site (17). Perhaps because of
the requirement of its flexibility in enzyme catalysis, this loop was
invisible in the previous crystal structure of the apoenzyme. Neither was this
loop observed in the crystal structure of UPPs from M. luteus, in
which both subunits had the closed conformation
(10).
To answer the questions regarding the location of substrate binding, the role of the metal ion, and the function of the flexible loop in enzyme catalysis, we report here the structure of UPPs with sulfates, Mg2+ ions, and two molecules of Triton X-100 occupying the tunnel in conjunction with the kinetic results of a few relevant mutants. Triton at low concentration has been shown to increase the UPPs steady-state reaction rate (18). However, a high concentration of Triton in the crystallization condition resulted in the occupancy of Triton in the active site. Therefore, the dose dependence of Triton in altering the reaction velocity was also examined.
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EXPERIMENTAL PROCEDURES |
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Crystallographic AnalysisFor purification of enzymes, our previously reported protocol of using nickel-nitrilotriacetic acid column was followed (18). The purified wild-type UPPs was crystallized using the hanging drop set-up from Hampton Research (Laguna Niguel, CA), while attempts were tried to incorporate FPP into the crystal. In the end, 2 µl of mother liquid containing 0.01 M cobalt chloride, 0.1 M MES, and 1.8 M ammonium sulfate at pH 6.5 was mixed with 2 µl of protein solution consisting of 10 mg/ml UPPs, 2% Triton X-100, 5 mM MgCl2, and 660 µM FPP. The mixture was equilibrated against 200 µl of the mother liquid at 25 °C. Crystals started to appear within 10 days. This condition was different from the previous one for the Se-Met enzyme (11), but the crystals turned out to be isomorphous. Diffraction experiments on the UPPs crystal was carried out at 150 °C on beam line 17B2 of the National Synchrotron Radiation Research Center in Hsinchu, Taiwan. Data were processed and scaled by employing the program HKL2000 (19). For computational refinement, manual modification, and analysis of the crystal structure, the programs CNS (20), O (21), and CCP4 (22) were used.
Using 2.0-Å resolution data and the apoenzyme model that we solved
previously, but with all solvent and cofactor molecules removed, an initial
R-value of 0.52 was calculated, and it was reduced immediately to
0.39 after rigid body refinement. The Se-Met residues in the original model
were replaced with methionines, and subsequent energy minimization yielded an
R-value of 0.29 before calculation of the first Fourier map.
According to the density level of 1.5 , 327 water molecules were added
to the model, and the amino acid side chains were adjusted. Strong densities
in the active site tunnel of one subunit (monomer B) clearly showed two Triton
molecules, but they were modeled as a series of solvent atoms. Possible
densities for sulfate and metal ions were also modeled as water molecules.
Prior to subsequent refinement, 5% of randomly selected reflections were
set aside to calculate Rfree values for monitoring
progress of refinement (23).
The model yielded R and Rfree values of 0.227 and
0.256 after simulated annealing and temperature factor refinement when the
resolution was increased to 1.8 Å. Explicit models for the two Triton
molecules, the sulfate, and the magnesium ions were constructed, and the
polypeptide termini were also modified according to the Fourier maps. With the
bound cofactors and 393 water molecules, the R and
Rfree values were reduced to 0.200 and 0.231,
respectively. At this point, densities for residues 7282 of monomer B
became interpretable in the map, and the corresponding fragment was modeled.
Finally, the resolution was increased to 1.73 Å, and more water
molecules were added according to 1.0 density level in the
2Fo Fc map.
Statistical numbers for the diffraction data set and the refined model are
listed in Table I.
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Site-directed Mutagenesis of UPPsUPPs mutants were prepared
by using PCR techniques in conjunction with the E. coli Bos-12 UPPs
gene template in the pET32Xa/Lic vector. The mutagenic primers used were
prepared by MdBio, Inc. The mutagenic oligonucleotides for performing
site-directed mutagenesis are as follows: 5'-GCCTTTGGGGCTAAAGCC-3'
for H43A; 5'-GGGGAGGCTCGCATTAGT-3' for H199A; and
5'-GGGGAGGCTCGCATTAGT-3' and 5'-GCCTATGCCGCACTTTAC-3'
for the H199A/E213A double mutant. Subsequently, the forward primer
5'-GGTATTGAGGGTCGCATGTTGTCTGCT-3' and the reverse primer
5'-AGAGGAGAGTTAGAGCCATCAGGCTGT-3' were used in combination with
the PCR products obtained using the above mutagenic oligonucleotides to create
the full-length mutant UPPs genes. The FXa cleavage site (IEGR) and the
complementary sequences to the sticky ends of the linear vector pET-32Xa/LIC
were included in these primers. Thirty cycles of PCR were performed using a
thermocycler (Applied Biosystems) with the melting temperature at 95 °C
for 2 min, annealing temperature at 42 °C for 2 min, and polymerization
temperature at 68 °C for 40 s. The PCR product was subjected to
electrophoresis on 0.8% agarose gel in Tris-acetate-EDTA (TAE) buffer, and the
gel was then stained with ethidium bromide. The part of the gel containing the
band of the correct size was excised, and the DNA was recovered using a DNA
elution kit. The constructed gene of a mutant enzyme was ligated to the vector
by incubation for 1 h at 22 °C. The recombinant UPPs plasmid was then used
to transform E. coli JM109 competent cells that were streaked on a
Luria-Bertani (LB) agar plate containing 100 µg/ml ampicillin.
Ampicillin-resistant colonies were selected from the agar plate and grown in 5
ml of LB culture containing 100 µg/ml ampicillin overnight at 37 °C.
The mutation was confirmed by sequencing the entire UPPs mutant gene of the
plasmid obtained from the overnight culture. The correct construct was
subsequently transformed to E. coli BL21 for protein expression. The
5-ml overnight culture of a single transformant was used to inoculate 500 ml
of fresh LB medium containing 100 µg/ml ampicillin. The cells were grown to
A600 = 0.6 and induced with 1 mM
isopropyl--thiogalactopyranoside. After 45 h, the cells were
harvested by centrifugation at 7,000 x g for 15 min.
Measurements of Km and
kcat Values for Mutant UPPsFor the
measurements of kinetic parameters, mutant UPPs (0.01 µM H199A,
0.5 µM H199A/E213A, or 0.5 µM H43A) was utilized
to initiate the reaction of FPP and [14C]IPP in 200-µl
solutions. For IPP Km and
kcat determinations, 5 µM FPP was utilized
to saturate the enzyme, and IPP concentrations of 0.55-fold
Km were employed. For FPP
Km measurements, 0.220 µM
FPP were used along with 20 µM [14C]IPP. All
reactions were carried out in 100 mM KOH-Hepes buffer, pH 7.5, 50
mM KCl, and 0.5 mM MgCl2 at 25 °C in the
presence of 0.1% Triton X-100. To measure the initial rate, 40-µl portions
of the reaction mixture were periodically withdrawn within 10% substrate
depletion and mixed with 10 mM EDTA for reaction termination. The
radiolabeled products were then extracted with 1-butanol, and the
radioactivities associated with aqueous and butanol phases were separately
quantitated by using a Beckman LS6500 scintillation counter. Initial velocity
data were fitted to Equation 1 to
obtain Km and kcat values by
non-linear regression using KaleidaGraph computer program. The
kcat was calculated from
Vmax/[E], as shown in
Equation 1,
![]() | (Eq. 1) |
Enzyme Activity Measurement under Different Concentrations of Triton X-100 To measure the kinetic constant of E. coli UPPs in the presence of Triton X-100, the enzyme reaction was initiated by adding 0.1 µM UPPs to a reaction mixture containing 0.001% Triton X-100, whereas to reaction mixtures containing 0.01, 0.05, 0.07, 0.1, 0.5, or 1.5% of Triton X-100 (v/v), 0.01 µM enzyme was added. All reactions were carried out in 5 µM FPP and 50 µM [14C]IPP along with 100 mM Hepes-KOH buffer (pH 7.5), 50 mM KCl, and 0.5 mM MgCl2. Portions of the reaction mixture were periodically withdrawn within 10% substrate depletion and mixed with 10 mM EDTA for reaction termination. The radiolabeled products were then extracted with 1-butanol, and the radioactivities associated with aqueous and organic phases were separately quantitated by using a Beckman LS6500 scintillation counter. Under the saturated concentration of FPP and IPP (>5-fold Km value), the rate of IPP condensation was determined as kcat (s1).
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RESULTS AND DISCUSSION |
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In contrast, monomer A remained largely unchanged as in the apo-UPPs
crystal. Only slight shifts were observed in the helices 3 and
4, with maximal displacements of 1 Å, although the kink of helix
3 seems to occur at Glu-96 also rather than at Ala-92, as in apo-UPPs.
Apparently, monomer A assumes a closed conformation, as in the apoenzyme. The
B-values of residues 8896 are between 40 and 60
Å2, whereas those of 8687 are over 60
Å2, indicating some disorder in this region. Other regions
with B-values higher than 40 Å2 are located in
residues 114115 of monomer A and 7481 of monomer B,
corresponding to the loops of
C-
4 and
B-
3,
respectively. Movement of these two helices, which constitute the forewing in
the butterfly-shaped UPPs molecule
(11), accounts for the
conversion between open and closed conformations and, thus, modulates binding
and release of the substrate and product from the tunnel-shaped active
site.
Adjacent to the flexible loop, residues 6971 at the C terminus of
strand B show maximal displacements
(Fig. 1C).
Specifically, the entire residues of Ala-69, Phe-70, and Ser-71 in monomer B
were moved by 3.3, 5.8, and 7.5 Å, respectively. The phenyl group of
Phe-70 was moved by 14.0 Å from the original position in apo-UPPs, where
it was only <2 Å away from a bound sulfate ion in the current
structure. Such structural changes presumably were a result of the bound
sulfate ion and Triton molecules in the active site. It now interacts directly
with the tert-octyl phenyl head group of Triton (see below). The
neighboring side chains of Leu-85, Leu-88, Phe-89, Trp-91, and Ser-95 in the
3 helix were also rotated to accommodate the Triton molecule. Similar
rearrangements for residues 6971 were also observed in monomer A, but
the side chains in helix
3 remained in their original positions,
perhaps because of the absence of the Triton molecule. The N-terminal part of
helix
3 that encompasses residues 7985 and the connecting loop
of 7278 to strand
B were not observed in monomer A. Presumably,
these residues were flexible, as in the apoenzyme.
The Sulfate IonsBoth FPP and IPP substrates, as well as the reaction intermediates and the final product UPP, have a highly negatively charged pyrophosphate moiety in the molecule. In the previously solved crystal structure of farnesyl pyrophosphate synthase, the allylic and homoallylic substrates are bound via Mg2+ions, which are coordinated with the active site Asp residues in the conserved DDXXD motifs (9). However, a similar DDXXD motif was not found in cis-prenyltransferases. In contrast, the crystal structure of UPPs from M. luteus (10) showed a sulfate ion bound to a highly conserved structural P-loop that contains the positively charged Arg-32 (equivalent to Arg-30 in the E. coli enzyme). This structural P-loop is supposed to interact with the pyrophosphate of the allylic substrate FPP. A second binding site for pyrophosphate of the homoallylic substrate IPP was also proposed (10, 16). It involves two positively charged arginine residues, 197 and 203 (Arg-194 and Arg-200 in E. coli). In both cases, Mg2+ ions were also supposed to participate in substrate binding by bridging the pyrophosphates of FPP and IPP with the acidic side chains of Asp-29 and Glu-216* (Asp26 and Glu213* in E. coli).2
In our refined UPPs structure there are five sulfate ions, and both monomers have two sulfates bound in the active site, as shown in Figs. 1 and 2. The first sulfate ion (S1) forms five well defined hydrogen bonds with the backbone nitrogen atoms of Gly-27, Gly-29, and Arg-30 and the side-chain NE and NH2 atoms of Arg-30. The side chain of the less conserved Arg-39 is 2.8 Å from the S1 sulfate in monomer A, but the distance is 4.0 Å in monomer B. Nevertheless, this Arg-39 as well as the Arg-77 in the flexible loop may both participate in substrate binding and charge neutralization in case of a more negative pyrophosphate. The second sulfate ion (S2) forms four hydrogen bonds with the side-chain nitrogen atoms NH2 of Arg-194, NE and NH2 of Arg-200, and the oxygen OG1 of Ser-202, which is hydrogen bonded to NH1 of Arg-194. One of the S2 oxygen atoms in monomer A is 2.7 Å from the backbone N of the C-terminal Arg-242*, indicating a fifth hydrogen bond. The side chain of Arg-241* is 3.4 Å from the S2 ion. Again, these two arginines and the nearby Arg-239* may interact with the pyrophosphate group of the substrate, although none of them is strictly conserved.
A fifth sulfate ion was observed on the outer surface of monomer B (see
Fig. 1C). It is bound
directly to the side chain of Arg-102, forming two well defined hydrogen bonds
with the NH1 and NH2 atoms. Two additional water-mediated hydrogen bonds were
also observed between this sulfate and the backbone oxygen atom of Ser-95. It
may also interact with the nearby side chains of Lys-98 and a symmetry-related
Lys-33, but no direct bonds were seen. One of the two mediating water
molecules was also hydrogen-bonded to the backbone nitrogen atom of Ser-99 and
a third water molecule, which was bound to the backbone oxygen and nitrogen
atoms of Asp-94 and Lys-98, respectively. Insertion of these two water
molecules between the normally hydrogen-bonded backbone atoms actually
produced the kink at Glu-96 in the 3 helix. Similar intercalation of
waters in the
3 helix was also observed for monomer A, but the
densities were weaker.
Compared with the original model of apoenzyme, quite a few rearrangements
in the UPPs structure occurred upon sulfate binding. In monomer A, the side
chain of Arg-30 remains in original position, but the guanidium group was
flipped over to bind the S1 sulfate ion. Originally, it was hydrogen bonded to
Asp-26. The side chain of Asp-26 was rotated 120° for the
1 angle upon binding sulfate, and the carboxyl group became
bonded to the side chain of Arg-194 (Fig.
2B). The side chain of Arg-200 formed a salt bridge with
Glu-213* in apo-UPPs. In the present structure, the guanidium group was moved
4 Å upon swinging the side chain by 120° for the
1
angle to interact with the S2 ion. The side chain of Arg-39 also underwent
slight reorientation. In monomer B of the apo-UPPs, the side chain of Arg-30
had a different conformation, which did not interact with Asp-26. Upon sulfate
binding, it moved 5 Å toward the S1 ion by rotating almost 180°
about the
3 angle and made identical interactions with the
sulfate as in monomer A (Fig.
2A). The side chain of Asp26 in monomer B was also
rotated 120° and made hydrogen bonds with Arg-194. The side chain of
Arg-200, originally bound to Glu-213* and Glu-240*, was moved 5 Å toward
the S2 ion by 120° rotation about the
3 angle. Arg-39
remained almost unchanged, as in monomer A.
Consistent with the model proposed for the UPPs from M. luteus, the S1 and S2 sulfate ions observed in our crystal structure may represent the locations of the pyrophosphate groups of the allylic and homoallylic substrates FPP and IPP, respectively, in E. coli UPPs. However, no magnesium ion was observed to make direct interactions with the bound sulfate ions. As shown below, the magnesium ions are bound in other places, and they function in a different way than those in FPPs or other enzymes with pyrophosphate substrates.
The Magnesium Binding SitesThe role of the metal ion has often been argued in metal-requiring enzymes. In prenyltransferases, the common mechanism may be that Mg2+ chelated by Asp residues coordinates with the pyrophosphate moiety of substrate FPP and facilitates the nucleophilic attack by making the pyrophosphate a better leaving group. The trans-type prenyltransferases all have two DDXXD motifs responsible for allylic substrate (FPP) and homoallylic substrate (IPP) binding (24). The Asp residues in the motif play essential role in Mg2+ binding, and the substitution of these Asp residues with Ala led to the remarkable decrease of substrate affinity and turnover number (25, 26). However, none of these motifs is found in the cis-type enzymes. In the UPPs from E. coli, a possible candidate for binding Mg2+ in the active site is Asp-26 (or Asp-29 in the M. luteus enzyme). As shown above, this residue did have significant conformation change of the side chain upon binding sulfate, but no Mg2+ ion seemed to be involved. Nevertheless, it remains unanswered whether Mg2+ will participate in binding if the anions are actually pyrophosphates.
In the refined structure of UPPs crystallized in the presence of 5 mM MgCl2, there are two Mg2+ ions bound to the enzyme (Fig. 1). The two Mg2+ binding sites are equivalent. They are located in the dimer interface and related by the molecular dyad axis. As shown in Fig. 2C, each ion is octahedrally coordinated with six ligands; one of them is the ND1 atom in the side chain of His199, another is the OE1 atom of Glu213*, and the other four are water molecules. The water molecules are directly hydrogen bonded with the backbone O of Gly197, the side chain OE1 of Glu198 and the backbone N of His-199 in one subunit, as well as the side chain OE2 of Glu213* and the backbone O of Ala235* in another subunit. The hydrogen bond network is further extended with the involvement of ordered water molecules in this region. The Mg2+ binding site is 11 Å from the S2 site for sulfate ion in both monomers, and there is no direct interaction between the bound cation and anion.
In the previous structure of apo-UPPs, the C termini of both subunits were disordered, wherein no densities were observed beyond residue 240 of monomer A and residue 238 of monomer B. In the current structure, the C termini are also disordered. However, some notable rearrangements were observed. In both subunits, the imidazole ring of His-199 was rotated 90° into a proper orientation for coordinating the Mg2+ ion (Fig. 2C). Without the cation, the Mg2+ binding site was occupied by the positively charged side chain of Arg-239*. It was moved 11 Å to the other side of peptide backbone in the presence of Mg2+ ion and redirected toward the S2 sulfate ion in the active site. The side chain of Glu-240* was salt bridged with that of Arg-200 in the apo-UPPs, but in the current model the backbone atoms were displaced by 4.5 Å, and the side chain also moved 10 Å away from the active site, facing the solvent. Similar conformations of Arg-239 and Glu-240 are seen in both monomers, whereas the additional Arg-241 of monomer B is 3.4 Å from the S2 ion in monomer A. As shown above, Arg-200 is directly bonded to the S2 sulfate ion, while Arg-239* and Arg-241* may also be involved. Consequently, the binding of Mg2+ is likely to generate a more ordered structure of the C terminus for interactions with the pyrophosphate substrate.
Effects of Mutants on the UPPs ActivityAs shown in Table II, our results from a previous mutagenesis study showed that substitution of Asp-26 in E. coli UPPs by alanine decreased the kcat to only one-thousandth (103) of that for the wild-type enzyme without significant change of the Km values for FPP and IPP (14). Therefore, Asp-26 is important for catalysis but not for substrate binding. The IPP condensation mechanism of the enzymes for polyprenyl pyrophosphate synthesis has been well established (5, 6). A carbocation is first generated by eliminating the pyrophosphate in the allylic substrate, with the assistance of charge neutralization by Mg2+ or protonation of the leaving group. A proton on the second carbon of the homoallylic substrate is then subtracted prior to its nucleophilic attack on the allylic carbocation. The carboxyl group of Asp-26, located between the two bound sulfate ions in the active site, is a good candidate to accept proton from the substrate IPP during catalysis.
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Although it is uncertain whether Mg2+ ions participate in the catalysis by UPPs, the imidazole group of His-43 seems to serve as a proton donor to the pyrophosphate. It is located between the S1 sulfate ion and the Triton molecule, which represent the pyrophosphate and the hydrocarbon moieties of the substrate, respectively. As shown in Table II, the mutation of His-43 to Ala decreased the kcat to only one thousandth (103) of the original value. A moderate increase of the Km values was also observed. As a proton donor, the imidazole group should be protonated, and, because of the positive charge, it is likely to also participate in substrate binding.
Regarding the role of Mg2+ in substrate binding, our fluorescence studies showed that the FPP substrate still binds to UPPs and quenches its intrinsic fluorescence even in the absence of Mg2+ (17). However, the IPP binding absolutely requires Mg2+. As discussed above, binding of Mg2+ resulted in significant structural changes in the C-terminal regions of the enzyme, which allowed side chain rotations of Arg-200 and Arg-239* into a proper orientation for binding the S2 sulfate ion. Previous studies showed that Glu-213 is involved in IPP binding, because replacement of Glu-213 with Ala resulted in a significant 70-fold increase of IPP Km value and a 100-fold reduction of kcat (14, 16) (Table II). In the current structure, His-199 and Glu-213* constitute two ligands for binding Mg2+, and we further examined the importance of His-199 by mutating it to Ala. As listed in Table II, the mutant H199A shows a four times larger Km value for IPP. However, its role seems not as important as that of Glu-213. The His-199/Glu-213 double mutant also displays 70-fold larger IPP Km and 1,000-fold smaller kcat values. Therefore, Glu-213 is essential in binding Mg2+, but His-199 is optional. These results are consistent with the previous observation that both carboxyl oxygen atoms in the side chain of Glu-213 contribute to Mg2+ binding. On the other hand, in all three mutants the Km values for FPP remained nearly unchanged, providing further evidence for the distinction between S1 and S2 site in binding FPP and IPP.
Structures of the Bound Triton MoleculesIn the refined structure of UPPs studied here, no FPP molecule was observed in the active site, although the crystallization condition included a significant level of the substrate. Instead, two molecules of Triton X-100 were clearly seen in the electron density maps. These are shown in Fig. 3. The first Triton molecule (T1) has 24 non-hydrogen atoms with an extended conformation for its PEG moiety. The second molecule (T2) has 30 non-hydrogen atoms with the PEG moiety folded back on itself, resulting in a circular shape of the molecule. As shown in Fig. 1, both Tritons are bound to the active site tunnel of monomer B, which has an open conformation. The T1 molecule occupies the lower or inner part of the tunnel, whereas the T2 molecule binds to the upper or outer part, and it appears to block the opening of the tunnel. In the tunnel of monomer A there were elongated densities probably corresponding to fragments of other Triton molecules, but the densities were not clear enough for model building, although the first PEG fragment was observed in this monomer A of the apo-UPPs crystal (11).
Interaction between the Triton molecules and the active site residues of
UPPs are mostly hydrophobic, as expected. The "head" moiety of
Triton consisting of a phenyl ring and a tert-octyl group is entirely
hydrophobic. In the T1 molecule, it interacts with the side chains of the
Ala-47, Val-50, Val-54, Ala-92, Leu-100, Leu-107, Leu-139, and Ile-141 of
UPPs. Details of the hydrophobic interactions are listed in
Table III. Near the opening of
the tunnel, the head group seems to be penned up by a salt bridge between
Arg-51 and Glu-96 from the helices 2 and
3, respectively, which
constitute part of the tunnel wall. The amphipathic PEG tail of T1, for which
the present model contains three ethylene glycol units, is half-exposed to the
solvent. The distal part extends away from the tunnel beyond the opposing side
chains of Ser-55 and His-103 near its bottom. The head group of the second
Triton molecule, T2, also makes direct hydrophobic interactions with that of
T1 where the minimal distance between them is 4 Å. It also interacts
with the side chains of Ala-69, Phe-70, Phe-89, Ala-92, Leu-93, Ile-109,
Phe-116, Leu-120, Ile-124, Ile-141, and Ala-143 in the hydrophobic cleft of
the active site (Table III).
Above this hydrophobic cluster, two adjacent side chains of Trp-75 and Leu-85
tend to form a lid to cover the active site cleft. The PEG tail makes a U-turn
at the second ethylene glycol unit, which is opposed by Met-25 and Trp-221.
The third unit is in contact with Gly-46 and Val-50, and it is close to the
side chain of His-43, with a distance of 4.5 Å. The 4th and
5th ethylene glycol units fold back on the phenyl head group, and
they also interact with the tert-octyl group of the T1 molecule as
well as the side chains of Phe-89 and Ala-92. The remaining 45 units of
the PEG tail protruded out of the active site and were not observed because of
disorder.
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The hydrophobic nature of the Triton molecules and their propensity to replace the natural substrate FPP in binding to the active site cleft suggest that the Triton structure should somehow resemble the reaction intermediates or product of UPPs, as shown in Fig. 3, B and C. Because of the different stereochemistry (cis) UPPs catalyzes from that (all-trans) of substrate FPP, the product UPP likely adopts a folded structure. The length of the observed PEG tail of the T1 molecule is comparable with that of a straightened all-trans farnesyl group of the substrate, whereas the remainder of T1 together with the entire T2 molecule has about the same size of the cis-polyprenyl moiety of the product. Thus, the linear part the T1 and the other parts of Triton molecules mimics the trans- and cis-prenyl parts of the product, respectively. In addition, the shortest distance between the Triton molecules and the sulfate ions S1 and S2 are 7.6 and 12.4 Å, respectively. Consequently, it is more likely for the S1 site to serve for binding to the pyrophosphate moiety of the allylic substrate FPP.
High Concentration of Triton Inhibits the Enzyme Activity Our previous studies have shown that Triton X-100 at a low concentration of 0.1% increased the steady-state kcat of E. coli UPPs reaction by 190-fold (18). The rationale for this activity stimulation by Triton can be attributed to its ability to provide the preferred interaction with the product, which is highly hydrophobic, and thus facilitate product release from the active site, which is the rate-limiting step of steady-state catalysis. In the present structure, two Triton molecules were found to occupy the UPPs active site and prohibit binding of the natural substrate FPP. Therefore, we determined the UPPs activity in the presence of high concentration of Triton X-100 as used in the protein crystallization experiments. As shown in Fig. 4, the enzyme activity is increased with the addition of a low concentration of Triton, which is similar to the previous results. However, when the concentration is higher than 1%, the UPPs activity drops. Presumably a high concentration of Triton increases the chance for Triton to occupy the active site, converts most UPPs molecules into the full-open conformation, and thus inhibits the enzyme reaction.
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Active Site Structure and Catalytic MechanismIn the current
crystal structure of UPPs, the sulfate ions show the locations of
pyrophosphate binding sites for substrate. The Mg2+ ions
induce conformational changes in the C-terminal region to facilitate binding
of IPP. The two Triton molecules in the active site cleft may represent the
trans- and cis-prenyl moieties of the product. In a
plausible mechanism of UPPs, the deprotonated IPP acts as a nucleophile to
attack the carbocation formed by FPP with the release of its pyrophosphate
group, similar to other prenyltransferase reactions
(27). As suggested by our
structural analysis, Asp-26 and His-43 play key roles in the enzyme reaction
in which Asp-26 acts as general base to deprotonate IPP and His-43 provides a
proton to FPP for the dissection of its pyrophosphate group. Both of their
mutations to Ala resulted in a significant decrease of
kcat (Table
II). The loop of B-
3 containing Glu-73, Trp-75, and
Arg-77 may also be involved in the catalysis. Our previous site-directed
mutagenesis studies have shown that the mutation of these three residues to
Ala led to lower substrate affinity and catalytic activity
(11). An overview of the
active site structure with the proposed reaction mechanism for UPPs catalysis
is shown in Fig. 5.
The subunit conformations with the primary substrate analogue of sulfate
(monomer A) and the additional product analogue of Triton (monomer B) are
different, particularly in the 3 helix. The interconversion between two
conformations of open and closed forms is implicated in substrate binding and
product release. The conformational change during substrate binding and
catalysis of UPPs has been probed previously using steady-state and
stopped-flow fluorometers
(17). It was shown that FPP
binding quenches the fluorescence of Trp-91 in the
3 helix, which moves
toward the active site during substrate binding and thereby results in a
closed conformation to provide better interaction of UPPs with the substrate.
After the reaction, the crowding prenyl chain of the product shifts the UPPs
structure to an open conformer for product release.
As a summary, the UPPs turnover is shown in Fig. 5D. The binding of FPP followed by an incoming IPP initiates the condensation reaction. The condensation occurs with the release of pyrophosphate from FPP, leading to addition of five carbon atoms to the growing hydrocarbon chain. A similar reaction is repeated by incorporating another IPP molecule, and it proceeds until the FPP chain elongation yields the C55 final product. The all-trans C15 portion of the product reaches the bottom of active site cleft, which was represented by the linear Triton molecule (T1). The other curved Triton (T2) resembles the cis-prenyl portion of the product.
The UPPs structure studied here shows two conformations; monomer A is in the "closed" form because it does not contain a well defined Triton or PEG molecule bound in the active site, and monomer B is in the "full-open" form because two product-like Triton molecules are bound. However, in the structure of apo-UPPs, monomer B has an "open" conformation although the active site is empty. Interestingly, another UPPs crystal contains a bound Triton in the active site of monomer A, which also has a closed conformation.3 Thus, the closed form of monomer A, and the open and full-open forms of monomer B probably correspond to "start," "idle," and "stop" status, respectively. Although the loop of 7282 can be seen in the present UPPs crystal, the precise functions of the strictly conserved residues in this region remain undetermined. To visualize other conformations and fully elucidate the catalytic process, further crystallographic work on UPPs complexes with various substrate analogues is in progress.
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FOOTNOTES |
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* This work was supported by National Science Council Grant
NSC91-2113-M-001-007 (to P.-H. L.) and a grant from Academia Sinica (to A.
H.-J. W.). 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.
These authors contributed equally to this work.
To whom correspondence may be addressed. Tel.: 886-2-2785-5696 (ext. 6070);
Fax: 886-2-2788-9759; E-mail:
phliang{at}gate.sinica.edu.tw.
¶ To whom correspondence may be addressed. Tel.: 886-2-2788-1981; Fax: 886-2-2788-2043; E-mail: ahjwang{at}gate.sinica.edu.tw.
1 The abbreviations used are: UPPs, undecaprenyl pyrophosphate synthase; UPP,
undecaprenyl pyrophosphate; IPP, isopentenyl pyrophosphate; FPP, farnesyl
pyrophosphate; PEG, polyethylene glycol; MES, 2-morpholinoethanesulfonic
acid.
2 Residues in the counter subunit are designated by an asterisk after the
position number (e.g. Glu-216*).
3 S.-Y. Chang, T.-P. Ko, P.-H. Liang, and A. H.-J. Wang, unpublished
data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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