From the Instituto de Biomedicina de Valencia
(Consejo Superior de Investigaciones Científicas), C/Jaime Roig
11, 46010 Valencia, Spain and the
Centro de
Investigación y Desarrollo (Consejo Superior de Investigaciones
Científicas), C/Jordi Girona 18-26, 08034 Barcelona, Spain
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ABSTRACT |
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The hyperthermophiles Pyrococcus
furiosus and Pyrococcus abyssi make
pyrimidines and arginine from carbamoyl phosphate (CP) synthesized by
an enzyme that differs from other carbamoyl-phosphate synthetases and
that resembles carbamate kinase (CK) in polypeptide mass, amino acid
sequence, and oligomeric organization. This enzyme was reported
to use ammonia, bicarbonate, and two ATP molecules as
carbamoyl-phosphate synthetases to make CP and to exhibit
bicarbonatedependent ATPase activity. We have reexamined
these findings using the enzyme of P. furiosus expressed in
Escherichia coli from the corresponding gene cloned in a
plasmid. We show that the enzyme uses chemically made carbamate rather
than ammonia and bicarbonate and catalyzes a reaction with the
stoichiometry and equilibrium that are typical for CK. Furthermore, the
enzyme catalyzes actively full reversion of the CK reaction and
exhibits little bicarbonate-dependent ATPase. In addition,
it cross-reacts with antibodies raised against CK from
Enterococcus faecium, and its three-dimensional structure, judged by x-ray crystallography of enzyme crystals, is very similar to
that of CK. Thus, the enzyme is, in all respects other than its
function in vivo, a CK. Because in other organisms the
function of CK is to make ATP from ADP and CP derived from arginine
catabolism, this is the first example of using CK for making rather
than using CP. The reasons for this use and the adaptation of the
enzyme to this new function are discussed.
Two types of enzymes, carbamate kinase
(CK)1 and carbamoyl-phosphate
synthetase (CPS), are known to synthesize carbamoyl phosphate (CP) from
mixtures of ATP, bicarbonate, and ammonia. CK reversibly makes CP
according to the following reaction (1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
In this reaction one ATP molecule is used/molecule synthesized of
CP, and the true substrate that is phosphorylated is carbamate, which
is generated chemically from bicarbonate and ammonia (1-3). Because of
the unfavorable equilibrium of the reaction, CK is thought to function
in vivo exclusively in the direction of ATP synthesis using
the CP generated by catabolic ornithine transcarbamylase in the
fermentative catabolism of arginine (4).
(Eq. 1)
In contrast to CK, CPS synthesizes irreversibly the CP that is used in the biosynthesis of pyrimidines, arginine, and urea, according to the following reaction (5).
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CK and CPS also differ structurally. CK is a homodimer of a polypeptide
of approximately 33 kDa (6), whereas CPS is a 120-kDa polypeptide that
is either associated or fused to another polypeptide of approximately
40 kDa (7). Alignment of the amino acid sequences of CK and CPS failed
to reveal the existence of a statistically significant sequence
identity between the two enzymes (8), whereas there is a high degree of
sequence identity among different CKs (6) or different CPSs (9). No
obvious structural similarities are found when the recently determined
three-dimensional structures of CPS from Escherichia coli
(10) and of CK from Enterococcus faecalis (11) are compared.
The two proteins exhibit an open -sheet
/
structure. Whereas
CPS exhibits the fold found in biotin carboxylase and in other proteins
that synthesize acylphosphate bonds, the CK fold appears not to be
represented in structural data bases, although it is likely to be found
in other enzymes of presently unknown structure that synthesize
acylphosphates and exhibit sequence similarity with CK, such as
acetylglutamate kinase,
-glutamyl kinase, and long chain fatty
acyl-CoA synthetases (6).
Given the important differences between CPS and CK, the recent
description of CK-like CPSs in the hyperthermophilic archea Pyrococcus abyssi (12) and Pyrococcus furiosus
(13, 14) is puzzling. In the extracts of these extremophiles that live at 100 °C and, in the case of P. abyssi, at high pressure
in the ocean bottom, the CK-like CPS was the only activity found to
synthesize CP in reaction mixtures containing ATP, bicarbonate, and
ammonia (12-14). The polypeptide mass, homodimeric nature, and amino
acid sequence (reported only for P. furiosus) of these
pyrococcal enzymes (12, 14) are characteristic of CKs. However,
similarly to CPSs, these enzymes were reported (12, 14) to use two ATP molecules/molecule made of CP and to exhibit ATPase activity in the
absence of ammonia, although the magnitude of the ATPase was greater,
relative to the overall reaction, than in classical CPSs (15, 16). In
contrast with most CPSs, which use glutamine with preference to ammonia
as the nitrogen source (7), the pyrococcal CPSs use exclusively ammonia
(12-14) but this is also the case with the ureotelic CPSs (7). Given
these puzzling characteristics of pyrococcal CPS, we decided to study
it in detail as it might represent an intermediate step in the
evolution of CP biosynthesis (14). Thus, we have cloned and
hyperexpressed in E. coli the gene encoding the CPS from
P. furiosus, and we have purified and crystallized the
recombinant enzyme generated in E. coli. The large amounts
of pure protein obtained in this way have permitted us to study the
stoichiometry, reversibility, point of equilibrium, and the nature of
the substrates in the reaction. Our results unequivocally show that the
enzyme catalyzes the CK reaction. Furthermore, our initial results of
x-ray studies on enzyme crystals also indicate that the structure of
this enzyme resembles closely that of CK. Therefore, this appears to be
the first example of a CK with an anabolic role that is reserved in other organisms for CPS (the synthesis of CP as a precursor of arginine
and the pyrimidines).
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EXPERIMENTAL PROCEDURES |
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Materials-- Recombinant enterococcal CK was isolated from E. coli BL21 (DE3) cells (obtained from Novagen) transformed with the plasmid pCK41 exactly as described (6). The preparation and characterization of monoclonal antibodies mAbCK1, mAbCK2, which recognize epitopes within the C-terminal 14 residues of enterococcal CK, and mAbCK3, which recognizes an epitope toward the center of the polypeptide, have been reported already (6). Polyclonal monospecific antisera against enterococcal CK were prepared by immunization of rabbits with the purified recombinant enzyme following a standard immunization protocol (17). E. coli CPS was purified as described (18). Pyruvate kinase, lactate dehydrogenase (both from rabbit muscle, salt-free), and V8 staphylococcal protease were from Sigma. Ornithine transcarbamylase was purified partially, free of CK, from E. faecium according to Ref. 1. Hexokinase and glucose-6-phosphate dehydrogenase (both from yeast) were from Roche Molecular Biochemicals. Enzymes were freed from ammonium sulfate and were placed in the buffer used in the assays by centrifugal gel filtration through Sephadex G-50 (19). Buffer pH values were determined at 22 °C. Goat anti-rabbit IgG or anti-mouse IgG conjugated with peroxidase were from Promega, ammonium carbamate was from Aldrich, dimethyl suberimidate was from Pierce, and polyethylene glycols were from Fluka or Hampton. Other reagents were of the highest quality available.
Polymerase Chain Reaction Cloning of the P. furiosus Gene for
CPS--
Genomic DNA from P. furiosus (a generous gift of
Dr. F. E. Jenney, Jr., Dept. of Biochemistry, University of
Georgia, Athens, GA) was used as a template for polymerase chain
reaction amplification of the CPS gene using a high fidelity
proofreading thermostable DNA polymerase (Deep Vent, New England
Biolabs) and the primers 5'-GTGGTTTCCATGGGTAAGAGGGTAGTGATTGC-3' and
5'-GCATTCGCTAAGCTGGGTCTTCTAAAGTTCCTCAGG-3'. These primers were designed
to amplify the entire open reading frame for the CPS (14) and to
introduce a NcoI site at the initiator ATG and a
BlpI site downstream of the stop codon. The polymerase chain
reaction products were digested with NcoI and
BlpI and inserted into the corresponding sites of plasmid
pET-15b (Novagen) behind the T7 promoter using T4 DNA ligase (USB)
followed by transformation of E. coli DH5. The CPS gene
in the resulting plasmid called pCPS184 was sequenced using an ABII
prism DNA Sequenator (Applied Biosystems).
Expression and Purification of Recombinant P. furiosus
CPS--
E. coli BL21(DE3) cells transformed with the
plasmid pCPS184 were grown at 37 °C in a shaking incubator in 3 liters of LB broth containing 0.1 mg/ml ampicillin until an
A600 of 0.5 was reached. After a 3-h induction
with 1 mM isopropyl -D-thiogalactoside, the
cells (6 g) were harvested by centrifugation, resuspended in 40 ml of
50 mM Tris-HCl, pH 7.5, at 4 °C, and disrupted by sonication. The recombinant CPS was detected in a low amount. Therefore, the pCPS184-transformed BL21(DE3) cells were transformed with the plasmid pSJS1240 (a plasmid that incorporates the
spectinomycin resistance gene from pSJS974 (20) into the
SphI site of pRI952 (21)) (pSJS1240 was provided by Dr.
S. J. Sandler, Dept. of Microbiology, University of
Massachusetts), which allows the expression of the rare E. coli tRNA codons for arginine (AGA and AGG) and isoleucine (ATA).
A high degree of expression (about 15% of the protein) was observed in
these cells after overnight growth in LB medium containing 0.1 mg/ml
ampicillin and 0.05 mg/ml spectinomycin. Purification of the enzyme
from 10 g of cells obtained in this way was carried out at
0-4 °C as described for P. furiosus cells (14) except
for the omission of the final Sephadex G200, Superose P12, and Mono Q
column steps and the replacement of Blue Sepharose by Affi-gel Blue
(from Bio-Rad).
N-terminal Sequencing-- N-terminal automatic Edman sequencing was carried out on a Procise 494 gas-phase Sequenator (from Perkin-Elmer) at the Servicio de Química de Proteínas, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, using for sequencing excised bands from Coomassie Blue-stained Western blots in polyvinylidene difluoride membranes (Immobilon P, Millipore) (22).
Crystallization of P. furiosus CPS-- Crystallization conditions using the hanging drop vapor diffusion method were tested initially at 4 and 22 °C with the sparse matrix sampling procedure (23) by mixing 1.5 µl of protein solution (10 mg/ml in either 10 mM Tris-HCl, pH 7.5, or 50 mM Tris-HCl containing 20 mM ATP and MgCl2) and 1.5 µl of reservoir fluid. Crystals formed at 22 °C in about a week under a large range of conditions. The larger crystals obtained in the absence of ATP were produced in 15% polyethylene glycol 8000, 0.25 M Li2SO4 in 0.1 M Tris-HCl, pH 8.5. In the presence of MgATP, crystals reaching 0.7 mm were obtained in 1.3 M sodium citrate in the same buffer.
X-ray Crystallographic Studies--
For diffraction studies, a
harvesting solution was used consisting of, for crystals grown in the
absence of MgATP, 30% polyethylene glycol 8000, 5% ethylene glycol,
and 0.275 M Li2SO4 in 0.1 M Tris-HCl, pH 8.5, whereas, for crystals grown in the
presence of MgATP, 1.45 M Na citrate and 10% glycerol in
0.1 M Tris-HCl, pH 8.5, was used. Crystals of about
0.4 × 0.3 × 0.3 mm in size were examined with an Image
Plate (MAR RESEACH) area detector mounted on a Rigaku rotating copper
anode x-ray source ( = 1.5418 Å) operating at 40 kV and 100 mA. All
data were collected at 100 K temperature with crystals flash cooled
using the Oxford Cryosystem. Data sets were processed using the DENZO
and SCALEPACK programs (24). Molecular replacement was performed with
the AMoRe program (25) using polyalanine models of the structures of
enterococcal CK (11) and the N-terminal 315 residues of E. coli biotin carboxylase (Protein Data Bank, entry code: 1bcn) as
search models. The rotation and translation functions were performed
using amplitudes from 15 to 4.0 Å resolution. The values of the
rotation and translation top solutions using the CK monomer as search
model exceeded consistently the values of all other peaks: rotation
function peak height/noise, 7.5/5.4 (free enzyme) and 12.6/8.4
(ATP-bound); translation, correlation coefficient/noise, 49.3/23.4
(free enzyme) and 28.5/14.2 (ATP-bound); R factor/noise,
49/55.8 (free enzyme) and 54/57.6 (ATP-bound). The solutions were
evaluated graphically using the O program (26), resulting in the
consistent packing of P. furiosus CPS dimers in the crystal
without steric problems. A similar search done with biotin carboxylase
as a search model gave no clear solutions above the noise level.
Other Assays--
Indirect enzyme-linked immunosorbent assays in
96-microwell plastic plates (from Costar) were carried out as described
in Ref. 17 using phosphate-buffered saline solutions containing 2 µg/ml protein antigens and 2% bovine serum albumin with 0.1% defatted dry milk, respectively, for coating and blocking the wells.
Immunoperoxidase detection was carried out with
o-phenylenediamine as a chromogenic substrate (27). CP was
determined as Pi (28) after alkaline hydrolysis (29) or as
citrulline (30) by coupling with ornithine and ornithine
transcarbamylase. ADP and ATP were measured spectrophotometrically at
340 nm with pyruvate kinase/lactate dehydrogenase and
hexokinase/glucose-6-phosphate dehydrogenase, respectively (31).
Protein was measured according to Bradford (32) using a commercial
reagent from Bio-Rad and bovine serum albumin as a standard. SDS-PAGE
was carried out according to Laemmli (33). Cross-linking with dimethyl
suberimidate and SDS-PAGE of the covalent adducts was done according to
Davies and Stark (34).
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RESULTS |
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Expression and Characterization of Recombinant P. furiosus
CPS--
The expected band of approximately 34 kDa (exact mass deduced
from the gene sequence (14), 34.3 kDa) was detected by SDS-PAGE in low
quantity (<5% protein) in extracts of E. coli cells
carrying the pCPS184 plasmid but not in extracts of cells carrying the progenitor pET-15b plasmid without the insert (Fig.
1). The low expression appeared to be
because of inefficient translation caused by the frequent occurrence in
the pyrococcal gene of the AGA, AGG, and ATA codons for arginine and
isoleucine, three codons that are rarely used in E. coli and
that appear 25 times in the P. furiosus gene on six
occasions as three adjacent pairs (14). This interpretation was
confirmed by the drastic increase in the production of the recombinant
enzyme that was observed when the pCPS184-carrying E. coli
cells were transformed with plasmid pSJS1240 (20, 21), which encodes
the tRNAs for these rare codons (Fig. 1). The enzyme produced by the
doubly transformed E. coli cells was soluble and active and
was purified to >95% homogeneity (Fig. 1, right panel),
exhibiting a specific activity in the assay of Legrain et
al. (13) of 0.075 and 1 mmol CP · h1
· mg protein
1 at 37 and 60 °C,
respectively. The activity is similar, although somewhat higher than
that reported for the enzyme purified from P. furiosus cells
(14). The N-terminal sequence was confirmed to be GKRVVIALG, and thus
E. coli removes the initial methionine in a manner similar
to that observed in P. furiosus (14). Limited digestion with
V8 staphylococcal protease yields two complementary fragments of
approximately 23 and 13 kDa (SDS-PAGE estimate, Fig. 1), of which the
23-kDa fragment exhibits the N-terminal sequence of the intact enzyme,
and the 13 kDa fragment has the N-terminal sequence DGEIKGVEAV and thus
corresponds to the fragment that begins in residue 203 of the reported
amino acid sequence deduced from the gene sequence. In agreement with
the specificity of V8 staphylococcal protease, residue 203 is preceded
by a glutamate residue. The sequence-deduced mass of the fragment that
begins in residue 203 and ends in the C terminus of the enzyme is 12.4 kDa, which is in excellent agreement with the electrophoretic estimate.
All these data confirm the fidelity of the polymerase chain reaction
cloning strategy used to generate the recombinant enzyme and the
identity of the recombinant and naturally produced enzymes.
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The pyrococcal enzyme was recognized in enzyme-linked immunosorbent
assay tests and Western blots by polyclonal rabbit antiserum raised
against the CK from E. faecium, although approximately 50-fold higher concentrations of the antiserum were necessary to get
the same response as with E. faecium CK (Fig.
2). Of three monoclonal antibodies
against E. faecium CK only mAbCK1, recognizing an epitope
localized within the C-terminal end 14 residues of CK (6),
cross-reacted with the pyrococcal enzyme (data not shown), although
approximately 100-fold higher antibody concentrations were needed for
an equal reaction as with enterococcal CK. The immunological
cross-reactivity of the two enzymes reflects their relatedness; the
differences in reactivity being compatible with the 49% sequence
identity.
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Stoichiometry of the Reaction and ATPase Activity-- Table I compares the production of ADP, Pi, and CP by the pyrococcal enzyme with those observed with typical CK and CPS in an assay mixture containing ATP, bicarbonate, and ammonia. Because these assays also included large amounts of ornithine and ornithine transcarbamylase to convert the CP produced to citrulline and phosphate, CK and CPS should yield in these assays, respectively, 1 and 2 mol of both Pi and ADP/mole of citrulline produced. The results obtained with enterococcal CK and E. coli CPS agree, within experimental error, with these expectations. With the pyrococcal enzyme the results, at both 37 and 60 °C (the ornithine transcarbamylase used for coupling is stable at 60 °C) (see Ref. 1), are essentially the same as with CK: a ratio of approximately 1 is found between the amounts produced of Pi, ADP, and citrulline.
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Previously the CPSs purified from P. abyssi (12) and P. furiosus (14) were reported to exhibit an ATPase activity in the absence of ammonia and in the presence of bicarbonate, corresponding to half of the ATP consumption in the presence of ammonia. When we assayed this ATPase activity with the recombinant enzyme at 37 and 60 °C, we did not detect such activity (Table I) unless the concentration of enzyme used was vastly increased (data not shown), corresponding to similarly low activity as with enterococcal CK (0.3% of full activity (1, 6)). In contrast, the bicarbonate-dependent ATPase activity of CPSs typically represents 10% of full activity (15, 16).
Equilibrium of the Reaction--
The equilibrium of the reaction
catalyzed by CPS is fully displaced toward CP synthesis (see Ref. 5),
whereas with CK the value of the Keq (1)
predicts conversion of only a small fraction of the ATP to ADP at the
concentrations of carbamate expected to be present in the assay
mixtures used here (1). The results illustrated in Fig.
3 confirm for E. coli CPS and
enterococcal CK these expectations. With the former the amount of CP
(Fig. 3) produced increases linearly with the amount of enzyme even when a large fraction of the ATP is used up, whereas with enterococcal CK the production of CP rapidly flattens out with increasing amounts of
the enzyme, despite the existence of high concentrations of ATP and
large excesses of bicarbonate and ammonia assuring a constant concentration of carbamate. The results with the pyrococcal enzyme fully replicate those obtained with enterococcal CK, as expected if the
reactions catalyzed by the pyrococcal and enterococcal enzymes are
identical. Furthermore, the extent of the reaction with E. coli CPS monitored by the production of ADP (Fig.
4) was the same whether or not the CP
formed was removed by coupling with ornithine transcarbamylase, whereas
with both enterococcal CK and the pyrococcal enzyme, the addition of
ornithine transcarbamylase greatly increased the production of ADP, as
expected if the equilibrium were displaced in the forward reaction by
the removal of the product CP.
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Use of Carbamate as Substrate of the Pyrococcal Enzyme--
CK
phosphorylates carbamate (2, 3), whereas bicarbonate and ammonia are
the true substrates of CPS (5). In agreement with this, Fig.
5 shows that the production of CP
(determined as citrulline via coupling with ornithine transcarbamylase)
by E. coli CPS is the same with fresh and aged mixtures of
potassium carbonate and ammonium chloride, whereas with enterococcal CK substantially more citrulline is formed with the aged than with the
fresh mixtures; for in the latter, at the moment of addition to the
assay carbonate and ammonia have not yet equilibrated with carbamate
(3). The results with the pyrococcal enzyme are similar to those with
enterococcal CK, indicating that carbamate is also the substrate for
the enzyme from P. furiosus. However, to observe differences
with aged and fresh mixtures, the mixtures had to be diluted more in
the case of the pyrococcal enzyme than with enterococcal CK. This
observation suggests that the pyrococcal enzyme has a lower
Km for carbamate than the CK from E. faecalis.2
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Further proof of the use of carbamate by the pyrococcal enzyme was
obtained (Fig. 6) by comparing the
production of CP when either ammonium carbamate or ammonium carbonate
was abruptly added to mixtures at pH 9.5 and 10 °C containing ATP
and the enzyme. These conditions were used by Jones and Lipmann (2) to
demonstrate the use of carbamate by E. faecalis CK, because
at this pH and temperature the stability of carbamate is increased. Our
results confirm (2) for enterococcal CK and demonstrate for the
pyrococcal enzyme, which is also active and stable at pH 9.5 (data not
shown), the production of more CP with ammonium carbamate than with the carbonate. Again, as in the experiments with the fresh and aged mixtures reported in the previous paragraph, lower concentrations of
carbamate and carbonate had to be used with the pyrococcal than with
the enterococcal enzyme to demonstrate the differences. In summary, the
results with fresh and aged solutions of ammonium carbonate and the
results using carbonate or carbamate concur by showing that the
pyrococcal enzyme uses carbamate rather than bicarbonate and ammonia as
the substrate of the reaction.
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Phosphorylation of ADP by Carbamoyl Phosphate-- The established function in vivo of CK is to synthesize ATP from ADP and CP. In agreement with this function, enterococcal CK catalyzes faster the phosphorylation of ADP than the synthesis of CP (1). In contrast, CPSs do not catalyze the full reversion of their reaction of CP synthesis (5, 15, 35), although they catalyze, as a partial reverse reaction occurring at a rate of only 20% of the full reaction (5, 15), the synthesis of one molecule of ATP from one molecule of ADP and of CP. Table II confirms that enterococcal CK catalyzes faster the phosphorylation of ADP than the synthesis of CP. Under the conditions of the assays illustrated in the Table the rate of ATP formation by this enzyme is approximately 4-fold higher than the rate of CP synthesis. Table II also shows that the pyrococcal enzyme catalyzes actively, as expected for a CK, the formation of ATP from ADP and CP, although in this case the ratio between the forward and reverse reactions approximates unity, possibly reflecting the adaptation of this enzyme to the new function of making CP rather than using it.
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Structural Similarity with Enteroccal CK Revealed by X-ray
Studies--
Crystals of the pyrococcal enzyme grown in the absence or
presence of MgATP (Fig. 7) diffracted
with a conventional x-ray source to at least 2.6 and 2.0 Å resolution,
respectively, although spectra were collected at 2.9 Å (98%
completeness; Rmerge, 8.4) without ATP and at
2.2 Å (95.6% completeness; Rmerge, 11.7) with MgATP. The
space group is, for the crystal without substrates, tetragonal P4 with
unit cell parameters a = b = 97.78 Å and c = 135.42 Å. Packing density considerations (36)
indicate that for a monomer mass of 34.3 kDa the unit cell could
contain 16 monomers (Vm, 2.35 Å3/Da; solvent content, 47%), corresponding to four
monomers in the asymmetric unit. Cross-linking with dimethyl
suberimidate (Fig. 8) confirms (14) that
the enzyme is dimeric and reveals the formation of dimers of dimers.
Upon treatment with dimethyl suberimidate a major band appears
corresponding to the dimer and a less prominent band with the mass of
the tetramer is also seen, whereas the cross-linking of three monomers
is detectable but less frequent. Thus, a dimer of dimers is likely to
occupy the asymmetric unit of the crystal. Cross-linking of
enterococcal CK under the same conditions (Fig. 8) confirms its dimeric
character (1) and reveals lesser tendency to form dimers of dimers. In the presence of MgATP space group was orthorhombic
P212121 with unit cell parameters
a = 55.22 Å, b = 90.92 Å, and
c = 132.93 Å and an estimated number of 8 monomers/unit cell (Vm = 2.53 Å3/Da; solvent content, 51%) or two monomers, possibly
making a dimer, in the asymmetric unit.
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Given the existence of nearly 50% sequence identity between the
pyrococcal enzyme and enterococcal CK (6), and because we have recently
determined the three-dimensional structure of CK enterococcal by x-ray
crystallography (11), the molecular replacement method (37) was used to
attempt phasing of the observed structure factors from the coordinates
of enterococcal CK. For the spectra of the crystals grown in the
absence or in the presence of MgATP the results are unambiguous with
values of the rotation and translation solutions exceeding consistently
the values of all other peaks, which strongly indicates that the
correct orientation of the model was determined in the two cases (see
"Experimental Procedures"). The solution corresponds to dimers with
the same overall shape of the enterococcal CK dimer packed in the
crystal without interference between different dimers (Fig.
9). In contrast, a similar study made
with a polyalanine model of biotin carboxylase, which is the basic
structure of the catalytic domains of the typical, high molecular
weight CPS (10), failed to yield any unambiguous solution, indicating
that the folding of the CPS from P. furiosus resembles much
more CK than biotin carboxylase and, by extension, typical CPSs. In
addition, the organization of the monomers in the dimer given by the
solution obtained with the CK monomer does not resemble the subunit
organization in the quaternary structure of biotin carboxylase
(38).
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DISCUSSION |
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The present results clearly show that the CP-synthesizing activity previously reported in P. furiosus (13, 14) is due to an enzyme that uses chemically made carbamate and a single ATP molecule to synthesize CP reversibly. The reaction exhibits the characteristic equilibrium of the CK reaction, an equilibrium that does not favor at 37 °C the accumulation of CP. The enzyme catalyzes with comparable efficiency the forward and reverse reactions, and it is very inefficient in phosphorylating bicarbonate instead of carbamate, judging from its very small bicarbonate-dependent ATPase activity. All these properties are shared by typical CKs, such as the enterococcal enzyme (1, 2, 6). Our results are in conflict with the previously reported stoichiometry of 2 mol of ADP released/mole of CP formed by the partially purified enzyme from P. furiosus, assayed at 37 °C (14) or by the enzyme isolated from P. abyssi, assayed at 27 °C (12). Because these earlier enzyme preparations exhibited much greater ATPase activity in the absence of ammonia than the highly purified recombinant P. furiosus enzyme used here, the discrepancy would be explained if there were contaminating ATPases in the previous preparations that might have led to overestimation of the ADP production associated with CP synthesis. This possibility should be rigorously excluded with enzyme preparations obtained from cultures of pyrococci. Alternatively, it might be speculated that the highly purified enzyme used here is an individual component of a multicomponent CPS that would exhibit the classical CPS stoichiometry of 2 mol of ADP/mole of CP and that would be present in the possibly less pure preparations previously obtained from pyrococci (12, 14). However, this possibility is not supported by the similar specific activity and homodimeric nature of the recombinant and naturally produced enzymes and it also makes little biological sense, because the sole practical result of using such complex machinery would be to use an extra ATP molecule/mole of citrulline made in the ornithine transcarbamylase-coupled reaction. The coupling with ornithine transcarbamylase is essential in pyrococci given the rapid decomposition of CP at high temperature (13).
The high degree of sequence identity and immunological cross-reactivity of the pyrococcal enzyme and the enterococcal CK confirm the similarity of the two enzymes. Furthermore, our initial structural data obtained by x-ray crystallography clearly show that the pyrococcal enzyme exhibits a three-dimensional structure and quaternary organization that are highly similar to those of enterococcal CK and that are very different from those of CPS. Although the CK structure (11) reveals the existence of two catalytic sites/enzyme dimer, the relative orientation of the sites and the absence of intramolecular tunnels joining them exclude the possibility of catalytic collaboration between the two sites that is required for the synthesis of CP from bicarbonate and ammonia in three steps (bicarbonate phosphorylation, carbamate formation, and carbamate phosphorylation) that characterizes the mechanism of CPS (10, 39). In summary, all indicate that except for its extreme thermostability and low activity at normal temperatures,3 the pyrococcal enzyme is endowed with the characteristics of classical CKs. The finding of similar enzymes in P. abyssi (12) and Pyrococcus horikoshii (a gene has been found in this organism (GenBankTM accession number 3257702) encoding a putative polypeptide exhibiting 89% sequence identity with the enzyme from P. furiosus) strongly suggests that this enzyme constitutes a constant component of the catalytic machinery of the hyperthermophiles of the Pyrococcus genus.
As already indicated, the generally accepted function of CK is to
synthesize ATP from ADP and the CP produced mainly in the catabolism of
arginine by the arginine deiminase pathway (4). There are strong
reasons to deny such a function for the present CK. No arginine
deiminase activity was detected in the extracts of P. furiosus cells cultivated in a medium containing 0.2 g
arginine·liter1 (40), suggesting that the arginine
deiminase pathway is not operative in this organism. In keeping with
this, no arginine deiminase putative gene was identified in the entire
genome of the related organism P. horikoshii (41). If such a
gene had existed in P. horikoshii, it should have been
identified given the constant sequence motifs that are characteristic
of arginine deiminases (42). P. furiosus cells could be
grown in a defined medium containing ornithine as an arginine precursor
(40), and the enzymes of the biosynthetic pathway of arginine, anabolic ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinase were detected in this organism (43). Similarly, aspartate transcarbamylase, the enzyme that catalyzes the second step
of pyrimidine biosynthesis, was detected in both P. furiosus (13) and P. abyssi and was characterized in the latter
organism (44). Thus, CP has to be made in these microorganisms to be utilized by ornithine transcarbamylase and aspartate transcarbamylase in the biosynthesis of arginine and pyrimidines. The only CP-making activity detected in extracts from these microorganisms was due to the
enzyme studied here (12, 13). This enzyme appears to be coupled
functionally and to form physical complexes with ornithine transcarbamylase (13) and aspartate transcarbamylase (45) for there is
evidence of efficient channeling of the CP in the direction of
citrulline and carbamoyl aspartate biosynthesis. The inexistence in
these organisms of classical CPS is also supported by the lack of
detection of a classical CPS gene in the full genome of P. horikoshii (41). Again, it would be unlikely that such a gene
would have escaped detection because many constant regions of
characteristic sequence exist in all classical CPSs (9), and, for
example, classical CPS genes were detected (whereas no CK genes were
detected) in the genomes of the other three archea that have been
sequenced fully, Methanococcus jannaschii (46), Archaeoglobus fulgidus (47), and Methanobacterium
thermoautotrophicum (48). Taken together, all these data strongly
suggest that the CK studied here plays a new metabolic role: the
biosynthesis of CP for anabolic purposes.
Such an extraordinary use of a CK appears to be rendered possible by
the extreme living conditions of pyrococci. Whereas in the mesophilic
world the chemical formation of carbamate (49) might be slower than
required by the needs of CP, rendering essential the enzymatic
formation of carbamate by CPS in the initial two steps of its
reactional mechanism (39), the high temperature in hydrothermal vents
assures rapid chemical formation of carbamate without the need for
enzyme catalysis. Another function fulfilled by mesophilic CPS is the
provision of a high local concentration of carbamate at the site of
carbamate phosphorylation within the enzyme. This becomes possible by
coupling the synthesis of carbamate with the cleavage of an extra ATP
molecule. However, the enzymatic synthesis of high local concentrations
of carbamate appears unnecessary in P. furiosus given the
high affinity of the pyrococcal enzyme for carbamate,2
particularly because the concentration of carbamate may be relatively high in the habitat of P. furiosus given the finding in
hydrothermal vents of high concentrations of CO2 (50) (the
true reactant, rather than bicarbonate, in the chemical synthesis of
carbamate (49)) and of 0.6-1 mM ammonia possibly derived
from sediments (51) similar to those from where P. furiosus
was grown (52). In fact, the enzyme activity in P. furiosus
appears more than enough to serve the needs of arginine and pyrimidine
synthesis, even at suboptimal concentrations of carbamate. Thus, from
the increase in the enzyme activity given in Ref. 13, when the assay temperature is raised from 60 to 90 °C, the activity in the initial P. furiosus extract would be at 90 °C approximately 10 µmol · h1 · mg protein
1 (14), a value
that is 9-fold higher than the activity of CPS in E. coli
extracts, assayed at 37 °C (16). Another characteristic of the CPS
reaction that is mimicked by the high temperature of the habitat of
pyrococci is the irreversibility of the synthesis of CP, because the
rapid decomposition of CP at 100 °C (13) would cause the
concentration of this product to be essentially nil, thus displacing
strongly the reaction in the direction of CP synthesis. In summary, the
extreme living conditions of the pyrococci may render unnecessary the
enzymatic synthesis of carbamate by CPS with the associated expenditure
of an extra ATP molecule, as a prelude to making CP, explaining the use
of CK for CP synthesis in these organisms. It is of interest that CK
appears to have become adapted in P. furiosus to its new
anabolic function, because when compared with enterococcal CK, it
exhibits greater apparent affinity for carbamate2 and is
less effective in the synthesis of ATP from ADP and CP, relative to CP
synthesis. Other adaptations exhibited by the pyrococcal CK that
deserve further study are the much lower specific activity at
37 °C3 and much higher thermal stability than classical
CK. Detailed comparisons of the three-dimensional structures of the
enterococcal and pyrococcal CKs and site-directed mutagenesis of key
residues in the two enzymes will be essential to ascertain the reasons for these differences. Experiments with these goals are currently in
progress in our laboratory.
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ACKNOWLEDGEMENTS |
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We thank Drs. Francis E. Jenney, Jr. (Dept. of Biochemistry, University of Georgia, Athens, GA) for giving us genomic DNA from P. furiosus, S. J. Sandler (Dept. of Microbiology, University of Massachusetts) for providing pSJS1240, J. Cervera (Fundación Valenciana de Investigaciones Biomédicas, Valencia) for the gift of E. coli CPS and the monoclonal antibodies against CK, E. Grau (Instituto de Biología Molecular y Celular de Plantas, CSIC, Valencia) for automated DNA sequencing, and the Servicio de Química de Proteínas of the Centro de Investigaciones Biológicas (CIB-CSIC, Madrid) for N-terminal protein sequencing.
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FOOTNOTES |
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* This work was supported by Grants PM97-0134-CO2-01 and PB95-0218 of the Direccion General de Enseñanza Superior (DGES) of Spain.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.
§ Postdoctoral fellow of the Fundación Valenciana de Investigaciones Biomédicas-Bancaixa.
¶ Predoctoral fellow of the Generalitat Valenciana.
To whom correspondence should be addressed. Tel.: 34-963391772;
Fax: 34-963391773; E-mail: rubio{at}ibv.csic.es.
2 Preliminary estimates yield an approximate Km for carbamate of 7 µM. This value is about 10-fold lower than the Km for carbamate of enterococcal CK (1). The concentrations of carbamate were calculated from the equilibrium with bicarbonate and ammonia given in Ref. 1.
3
However, the activity at 100 °C of the
P. furiosus enzyme may be similar to that of enterococcal CK
at 37 °C. Thus, the pyrococcal enzyme released at 95 °C, under
the conditions of the CK assay (1), approximately 250 µmol
Pi·min1·mg protein
1,
whereas the enterococcal CK produces at 37 °C in the same assay approximately 600 µmol citrulline·min
1·mg
protein
1 (1).
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ABBREVIATIONS |
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The abbreviations used are: CK, carbamate kinase; CP, carbamoyl phosphate; CPS, carbamoyl-phosphate synthetase.
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REFERENCES |
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