Gain of Glutaminase Function in Mutants of the Ammonia-specific Frog Carbamoyl Phosphate Synthetase*
Amna Saeed-Kothe and
Susan G. Powers-Lee
From the
Department of Biology, Northeastern University, Boston, Massachussetts
02115
Received for publication, April 10, 2003
, and in revised form, May 7, 2003.
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ABSTRACT
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Depending on their physiological role, carbamoyl phosphate synthetases
(CPSs) use either glutamine or free ammonia as the nitrogen donor for
carbamoyl phosphate synthesis. Sequence analysis of known CPSs indicates that,
regardless of whether they are ammonia- or glutamine-specific, all CPSs
contain the structural equivalent of a triad-type glutamine amidotransferase
(GAT) domain. In ammonia-specific CPSs, such as those of rat or human, the
catalytic inactivity of the GAT domain can be rationalized by the substitution
of the Triad cysteine residue by serine
(1). The ammonia-specific CPS
of Rana catesbeiana (fCPS) presents an interesting anomaly in that,
despite its retention of the entire catalytic triad
(2) and almost all other
residues conserved in Triad GATs, it is unable to utilize glutamine as a
nitrogen-donating substrate
(3). Based on our earlier work
with the glutamine-utilizing E. coli CPS (eCPS), we have targeted
residues Lys258 and Glu261 in the fCPS GAT domain as
critical for preventing GAT function. Previously we have shown that
substitution of the corresponding residues in eCPS by their fCPS counterparts
(Leu
Lys and Gln
Glu) resulted in complete loss of GAT function
in eCPS (3). To examine the
role of these residues in the fCPS GAT component, we have cloned the
full-length fCPS gene from R. catesbeiana liver. Here we report the
first heterologous expression of an ammonia-specific CPS and show that a
single mutation of the frog enzyme, K258L, yields a gain of glutaminase
function.
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INTRODUCTION
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The evolution of a urea cycle that effectively removes excess, potentially
neurotoxic ammonia was critical for the adaptation of life to a terrestrial
rather than aquatic habitat
(46).
Arginine biosynthetic pathways were most likely the evolutionary precursors of
the urea cycle, with very few changes needed for pathway transformation
(6). Four of these changes
occurred in the enzyme that catalyzes the entry and rate-limiting step of the
urea cycle, carbamoyl phosphate synthetase
(CPS),1 and they were
as follows: (i) a decrease in Km for ammonia to
1 from
100 mM; (ii) a loss of interaction with glutamine
to avoid competition with the preferred substrate ammonia; (iii) localization
to the hepatic mitochondrial matrix to allow independent regulation and avoid
futile cycling; and (iv) gain of communication with a sensor of excess amino
acids, N-acetyl-glutamate (AGA, which serves as an essential
allosteric activator only for urea-synthesizing CPSs). In addition to the
ammonia-specific CPS required for urea synthesis, most organisms also express
a cytosolic glutamine-specific CPS that is involved in pyrimidine
biosynthesis. In Escherichia coli, a single, glutamine-specific CPS
(eCPS) participates in both arginine and pyrimidine synthetic pathways.
Glutamine-utilizing CPSs, e.g. eCPS, bind and cleave glutamine at
a glutamine amidotransferase (GAT) domain and channel the resulting free
ammonia, sequestered within the enzyme, to a synthetase (SYN) domain where all
other ligands are bound and all other reactions take place
(7,
8). Ammonia can substitute for
glutamine in eCPS, but with a much higher Km (111
versus 0.17 mM; Ref.
3). The contrasting properties
of ammonia-specific CPSs are even more intriguing when considered in the
broader context of the GAT family (comprising the Triad and Ntn subfamilies)
that participates in biosynthetic pathways for amino acids, amino sugars,
coenzymes, and purine and pyrimidine nucleotides
(9). Of the hundreds of GAT
family members characterized in various organisms and tissues, these CPSs are
the only enzymes that share the family-defining sequence motifs but have lost
the ability to utilize glutamine and gained the ability to scavenge low levels
of ammonia. For rat and human ammonia-specific CPSs, loss of glutamine usage
is explained, at least in part, by substitution of serine for the cysteine of
the catalytic triad (1,
10). However, the
ammonia-specific CPS of Rana catesbeiana (fCPS) retains the entire
catalytic triad (2) and almost
all of the other amino acids conserved in Triad GATs
(3) and, thus, is an ideal
candidate for detailed elucidation of the molecular basis for glutamine
discrimination in CPSs. Here we report that the present day frog
ammonia-specific CPS retains an unexpectedly close link to glutamine-utilizing
CPSs, with only a single mutation required for gain of glutaminase
function.
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EXPERIMENTAL PROCEDURES
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Strains, Plasmids, and Recombinant DNA MethodsThe ESP®
yeast protein expression and purification system, including the
Schizosaccharomyces pombe host strain SP-Q01 and Edinburgh minimal
medium, was from Stratagene. The S. pombe expression plasmid pESP-5
(11) was the generous gift of
Quinn Lu (GlaxoSmithKline). pCR2.1, used for cloning of PCR products, was from
Invitrogen. S. pombe transformations were carried out according to
the supplier (Stratagene). QuikChangeTM was used for site-directed
mutagenesis (Stratagene), and fidelity was verified by sequencing. Mutagenesis
primers (mutated codons set in boldface) were as follows for K258L, E261Q, and
K258L/E261Q, respectively:
5'-TTTGGCATCTGTCTCGGGAATGAAATTGCAGCTTTGGC-3';
5'-TTTGGCATCTGTAAAGGGAATCAAATTGCAGCTTTGGC-3'; and
5'-TTTGGCATCTGTCTCGGGAATCAAATTGCAGCTTTGCC-3'.
Cloning of Full-length fCPS cDNAR. catesbeiana total RNA
was prepared from frozen liver with the SV total RNA isolation system
(Promega) for amplification of full-length fCPS in reverse transcription PCR
reactions (RNA LA PCR Kit, Version 1.1; Takara Bio Inc.) with the supplied
oligo(dT)-adaptor primer and the gene-specific primer pair, respectively, as
follows: 5'-TTCGGCATATGAGCGTCAAGGC-3' (NdeI
site and start codon underlined); and
5'-ATAGTTCAGATCCCTAGGAGGG-3' (AvrII site and stop
codon set in boldface). The 4.4-kb fCPS amplicon was cloned into pCR2.1 (3.9
kb) to yield fCPS-pCR2.1, and the sequence of the entire gene was verified on
both strands. This cDNA encodes the mature fCPS protein (1463 amino acids)
with a methionine replacing the 33 N-terminal residues comprising the
mitochondrial matrix-targeting sequence. An NheI site (underlined)
was introduced at the 5'-end of the fCPS gene to allow subcloning an
NheI-BamHI fragment into pESP-5
(11), with
5'-TAAGAAGGAGCTAGCCATATGAGCGTCAAGGC-3' as the mutagenic
primer. The final expression construct encoded the mature fCPS protein fused
to an N-terminal His6-FLAG® tag
(HHHHHHDYKDDDKHASHM).
Purification of CPSNative frog liver
(12) and recombinant E.
coli CPSs (3) were
prepared as described previously. Recombinant wild type and mutant fCPS
proteins were expressed in S. pombe following an 1822-h
induction in 1-liter Edinburgh minimal medium according to the supplier's
protocol (Stratagene). Cells were harvested, resuspended in 1012 ml of
Buffer A (5 mM imidazole, 0.5 M NaCl, and 20
mM Tris, pH 7, 4 °C) containing 2 mM
phenylmethylsulfonyl fluoride and 0.1 mM each pepstatin, antipain,
leupeptin, chymostatin, and aprotinin, and transferred to a 50-ml BioSpec bead
beater chamber half-filled with pre-wetted, chilled glass beads (0.5-mm
diameter). The chamber was completely filled with glass beads, sealed, and
immersed in an ice, salt, and water slurry. Cells were lysed by bead beating
in 1-min pulses for a total of 35 pulses. The homogenate was rinsed out
of the chamber with Buffer A (plus protease inhibitors) in a total volume of
50 ml and clarified by centrifugation, followed by passage through a
0.450-µm Millex-HA (Millipore) filter. Cleared lysate (
50 ml) was
applied to a Hi-TrapTM chelating HP 5-ml column (ÄKTA FPLC, Amersham
Biosciences) charged with 0.5 M NiSO4 and equilibrated
in Buffer A. Bound protein was eluted from the column with a 040%
discontinuous gradient (010% in 25 ml, 1040% in 100 ml) of 0.5
M imidazole in Buffer A. The fCPS-containing fractions, eluting at
100150 mM imidazole, were pooled and concentrated by the
addition of solid ammonium sulfate to 90%. The protein was resuspended in
510 ml of Buffer B (10 mM Tris, 5 mM
MgCl2, 1 mM dithiothreitol, and 1 mM EDTA, pH
8.1), and loaded on a Hi-PrepTM 26/60 Sephacryl 200 column. fCPS was
eluted from this column in Buffer B, concentrated in Centriplus YM100
centrifugal filter devices to >5 mg/ml, and stored frozen at 80
°C. SDS-PAGE analysis indicated
BORDER="0"> 95% purity for all constructions.
Because protease treatments necessary to remove fusion partners also result in
proteolysis of fCPS, presumably at the links between domains
(1316),
in the present studies we retained the tag in all fCPS constructions.
Enzyme Assays and Data AnalysisCP synthesis was measured by
coupling the CPS reaction to that of ornithine transcarbamoylase and
quantitating the resulting citrulline
(3,
17). Glutamine-dependent and
ammonia-dependent ADP formations were determined in a pyruvate kinase/lactate
dehydrogenase-coupled assay as described previously
(3,
17). To determine
ammonium-dependent ADP formation, 30 mM NH4Cl was
included in the reaction mixture; to determine glutamine-dependent ADP
formation, 10 mM glutamine was included. Although only the
unprotonated form of ammonia is a substrate for CPSs
(18,
19), the data are presented as
the total of [NH4+] + [NH3] because it is the
level of NH4Cl that is varied during the experiments. Under the
conditions of the present studies, NH3 represents about 4% of the
total NH4+ added to the solution
(18). Glutamine hydrolysis was
determined by coupling glutamate formation to the glutamate
dehydrogenase-catalyzed reduction of 3-acetyl pyridine dinucleotide
(3). Kinetic data were fit by
non-linear regression (GraFit, version 5.1) to the equation
=
VmaxS/(Km + S),
where
is the initial velocity, Vmax is the maximal
velocity, S is the substrate concentration, and
Km is the Michaelis-Menten constant. Binding of
glutamine to CPSs was determined in a radiometric assay as described
previously (3).
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RESULTS AND DISCUSSION
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Cloning, Expression, and Characterization of Wild Type
fCPSDespite the critical role of ammonia-specific liver CPS, this
enzyme has not been as extensively studied as the glutamine-dependent CPSs,
primarily due to the absence of a recombinant expression system. Previously,
our laboratory has tried unsuccessfully to express ammonia-dependent CPSs in
several bacterial expression systems, with inclusion bodies resulting in all
cases. However, we have been able to express fCPS as a soluble, active protein
from the expression vector pESP-5
(11) in the fission yeast
S. pombe. Full-length fCPS was cloned from R. catesbeiana
liver and inserted in pESP-5 as described under "Experimental
Procedures." fCPS was expressed as the mature protein (1463 amino acid
residues), with the 33 N-terminal residues of the fCPS precursor replaced by a
methionine (set in boldface) and the His6-FLAG® fusion tag
(HHHHHHDYKDDDKHASHM). The N-terminal residues that were replaced serve
as a mitochondrial matrix-targeting signal and are normally cleaved as the
fCPS precursor crosses the inner mitochondrial membrane
(2). A two-step purification
protocol, with nickel affinity and size exclusion chromatography, yielded
1015 mg of pure soluble fCPS per liter of yeast culture medium. Because
our previous attempts to remove fusion partners by protease treatment revealed
additional proteolysis sites in fCPS, presumably at the links between domains
that are extremely susceptible to proteolysis
(1316),
we retained the tag in all fCPS constructions.
The kinetic and physical properties of recombinant wild type fCPS were very
similar to those observed for the native frog enzyme, indicating that the
structure of the recombinant protein mirrors that of the native protein and
further indicating that the fusion tag is a functionally neutral modification.
Recombinant and native fCPSs exhibited comparable ammonia-dependent CP
synthesis activities (1.17 and 1.12 µmol CP/min/mg, respectively), similar
kcat and Km values for
ammonia and ATP (Table I), and
both required the presence of the essential allosteric activator, AGA.
SDS-PAGE analysis confirmed that recombinant fCPS was the expected size (162
kDa), and recombinant and native fCPSs yielded essentially identical gel
filtration profiles (data not shown).
Gain of Glutaminase Function in fCPS MutantsBased on
analysis of GAT residues that are conserved in glutamine-utilizing CPSs but
not ammonia-specific CPSs (Fig.
1), we have identified Lys258 and Glu261 as
residues critical for preventing glutamine usage by fCPS
(3). Additional rationale for
targeting these residues was provided by our previous demonstration that
simultaneous occurrence of the corresponding substitutions in eCPS (Leu
Lys and Gln
Glu) prevent it from using glutamine
(3). Here we have constructed
in fCPS the reverse mutants K258L, E261Q, and K258L/E261Q. Both K258L and
K258L/E261Q could synthesize CP in the presence of glutamine, whereas E261Q
could not (Fig. 2). K258L,
K258L/E261Q, fCPS, and eCPS had similar rates for ammonia-dependent CP
synthesis (1.051.17 µmol/min/mg), whereas E261Q functioned at about
60% of this rate. AGA was required for CP synthesis by all fCPS constructs,
with either glutamine or ammonia as the nitrogen source. The absence of a
solved structure for fCPS prevents detailed structure/function analysis of the
mutants. However, our findings clearly demonstrate that the presence of lysine
at position 258 precluded use of glutamine and confirmed the critical role of
leucine at this position.

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FIG. 1. Comparison of invariant residues in the GAT domains of
glutamine-utilizing and ammonia-specific CPSs. Amino acid sequence
alignment (Munich Information Center for Protein
Sequences) of 23 different glutamine-utilizing CPSs, represented by
eCPS, revealed 29 residues that are invariant. Of these, 25 are also invariant
in ammonia-specific CPSs, including fCPS (R. catesbeiana), rCPS
(Rattus norvegicus), and hCPS (Homo sapiens).
Lys258 of fCPS, though not invariant, was included because its
polar character is very distinct from the nonpolar Leu or Met residues found
at this position in all glutamine-utilizing CPSs. The Cys-His-Glu catalytic
triad ( ) and the nine invariant residues (boldface) found in
all Triad GAT domains (9) are
also indicated.
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FIG. 2. Glutamine- and ammonia-dependent CP synthesis by fCPSs. Rates of
ammonia-dependent (gray bars) and glutamine-dependent (hatched
bars) CP synthesis were determined for native, recombinant, and K258L,
E261Q, and K258L/E261Q fCPSs in the presence of 30 mM
NH4Cl or 10 mM glutamine as described previously
(3). Comparison data for eCPS
(3) are included.
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To further define the interaction of the mutants with glutamine, we
directly measured glutamine hydrolysis
(Table II). Like other members
of the GAT family, eCPS can catalyze glutamine hydrolysis in the absence of
additional substrates (7),
although the rate of this uncoupled partial reaction is extremely slow
(kcat 0.24 min1; Ref.
20). This activity increases
dramatically (kcat 1.5 s1;
Table II) when glutamine
hydrolysis is coupled to CP synthesis on the SYN domain
(7) by the addition of ATP and
bicarbonate. Neither fCPS nor E261Q exhibited detectable hydrolysis or even
binding of glutamine under any of the conditions tested. When saturating
amounts of ATP and bicarbonate were present, both K258L and K258L/E261Q had
robust glutaminase activities (Table
II), although the kcat values were 7.5-fold
lower than that of eCPS. It should be noted that the eCPS glutaminase
kcat value of 1.5 s1 was
lower than the value of 3.44.7 s1
predicted by the ADP formation kcat values (6.8 and 9.4
s1; Table
II). Presumably, these kcat differences
reflect the different coupling systems used in the assays. The double mutant
exhibited a Km for glutamine that was about equal
to that of eCPS (0.20 versus 0.15 mM), whereas K258L had a
23-fold elevated Km for glutamine (3.45
mM), indicating that the E261Q mutation facilitated interaction
with glutamine. Surprisingly, given the well established effect on eCPS
behavior (7,
20), elimination of coupling
with CP synthesis had no effect on the glutaminase activity of K258L and
K258L/E261Q. When ATP was omitted from the glutaminase mix,
kcat values remained unchanged (0.2
s1 for both mutants), and the
Km values showed little change (6.57
mM for K258L and 0.74 mM for K258L/E261Q). This lack of
response to SYN substrates suggested that the fCPS mutants did not communicate
occupancy of the SYN active site to the GAT active site but, rather, had GAT
active sites that were permanently in the high activity conformation. It is
also note-worthy that, for both K258L and K258L/E261Q, AGA had no effect on
glutaminase activity.
Ability of fCPS Mutants to Synchronize Catalysis at the Multiple Active
SitesNext, we assessed the effects of the GAT mutants on SYN
domain function via ADP formation assays. Formation of the high energy
intermediate CP requires concomitant cleavage of two molecules of ATP to form
two ADPs (19,
21). In ammonia-dependent ADP
formation assays, K258L and K258L/E261Q displayed wild type kinetic parameters
for ammonia and ATP usage (Table
I). The E261Q mutation yielded modest changes in interaction with
ATP, possibly reflecting some long-range structural perturbation in this
mutant. In glutamine-dependent ADP formation assays
(Table II), we could not detect
any activity with the native, wild type, or E261Q fCPSs, whereas K258L/E261Q
did exhibit substantial ADP formation activity. The K258L/E261Q parameters
were consistent with those determined in the glutaminase assay, with the
Km for glutamine about equal to that of eCPS, and
the kcat being somewhat lower. Although K258L displayed
Michaelis-Menten behavior in the glutaminase assay and when ATP was the
variable substrate in the glutamine-dependent ADP formation assay, it failed
to do so when glutamine was the variable substrate in the latter assay
(Table II). Instead, production
of ADP by K258L was undetectable below a plateau glutamine concentration of
1 mM, and, as glutamine concentration was incrementally
increased to 20 mM, the rates measured did not show the expected
correlation with substrate concentration (i.e. the apparent
Vmax increased as the glutamine concentration ranged from
1 to 20 mM). This kinetic behavior suggested that the GAT domain of
K258L was acting independently of the SYN domain and was not coupling
glutamine cleavage to CP synthesis. The anomalous kinetic data further
suggested that the K258L GAT domain, rather than channeling the ammonia
sequestered within the protein, was releasing it into bulk solution so that CP
could be formed only when the solution ammonia concentration was equivalent to
that required for ammonia-dependent CP formation.
As an additional probe for synchronization between the GAT and SYN domains,
we determined the relative rates of product formation for fCPSs
(Fig. 3). When ammonia was the
aminating substrate, the production of CP and ADP increased steadily with time
of incubation and was consistently at or near the expected 1:2 ratio for wild
type fCPS and all three of the mutants. With glutamine as the aminating
substrate, the behavior of K258L and K258L/E261Q was markedly different, with
the production of CP lagging behind production of glutamate in ratios as high
as 1:13 and 1:10 for the double and single mutants, respectively. The ratio of
CP/glutamate became larger with increasing time of incubation but remained far
from the 1:1 ratio of a coupled system. Additionally, K258L and K258L/E261Q
formed excess ADP relative to CP, with respective ratios of 6:1 and 8:1 early
in the incubation and 3:1 ratios for both at 15 min. The uncoupling of ADP
formation from CP formation most likely reflects the nonproductive turnover at
the first ATP site that is known to occur when the intermediate carboxyl
phosphate reacts with water rather than ammonia
(1923).
Together, these findings indicated that both K258L and K258L/E261Q failed to
channel ammonia directly from the GAT to the SYN domain, thereby making CP
formation dependent on sufficient buildup of the ammonia released into
solution. The uncoupled character of K258L/E261Q was presumably masked in the
assay for glutamine-dependent ADP formation, whereas that of K258L was
apparent (Table II), because
the double mutant has a relatively low Km for
glutamine.

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FIG. 3. Relative rates of product formation in ammonia- and glutamine-dependent
reactions catalyzed by K258L and K258L/E261Q. fCPSs, including K258L and
K258L/E261Q, were incubated in a 1-ml volume for 5, 10, and 15 min at 37
°C, pH 7.6, in 50 mM HEPES, 50 mM NaHCO3,
10 mM ATP, 20 mM MgCl2, 10 mM KCl,
5 mM AGA, 1 mM dithiothreitol, 5 mM
ornithine, and ornithine transcarbamoylase (0.2 units, Sigma). At the
indicated times, 0.1-ml aliquots of the reaction mix were quenched and
neutralized, and the amounts of CP ( and ), ADP ( and ),
and glutamate ( ) were determined as described previously
(3).
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Potential Roles for the GAT DomainWe conclude that the GAT
domain must play a critical role in ammonia-specific fCPS, because it has been
retained so faithfully that a single mutation, K258L, was sufficient to enable
glutamine utilization. Occurrence of two simultaneous mutations (K258L/E261Q)
was even more effective, whereas the E261Q mutation conferred no detectable
gain of function, suggesting that the latter mutation provided compensatory
structural stabilization to the Triad scaffolding. The GAT domain of
present-day ammonia-specific CPSs might well serve an entirely structural role
but might also contribute to the lower Km for
ammonia relative to other amidotransferases. No ammonia site has yet been
identified for any GAT, nor has it been determined whether there is an
alternative to the tunnel for external ammonia entry to the SYN active site,
possibly sharing access with ATP and bicarbonate
(9,
20).
Our present findings clearly show that the cross-talk between the GAT and
SYN domains that occurs in eCPS was not established in the fCPS mutants K258L
and K258L/E261Q and that the ammonia derived from glutamine is not sequestered
within the tunnel. It is not yet clear how extensive the underlying changes
are (relative to a functional tunnel), whether they are confined to one or
both domains, or what path is taken by ammonia between the GAT and SYN active
sites. The molecular basis for coordination of the GAT and SYN active sites
connected by a channel has been elucidated for two other GATs, glutamine
phosphoribosylpyrophosphate amidotransferase
(24,
25) and imidazole glycerol
phosphate synthase
(2628),
and is based on a cycle of conformational changes that control access of
substrates, intermediates, and bulk solvent to the active sites and/or the
tunnel. Thus far, data for CPS are limited to a single solved conformation of
eCPS and identification of 10 GAT residues that appear to line the interior of
the tunnel (20). It is
noteworthy that fCPS has retained seven of these ten residues and has
conservative substitutions for the other three. Availability of a robust
expression system for fCPS, the first reported for any ammonia-specific CPS,
will greatly facilitate determination of the detailed molecular mechanism for
present-day CPSs and should also further elucidate the evolution of both the
CPS and GAT families.
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FOOTNOTES
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* This work was supported by National Institutes of Health Grant DK54423. 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. Tel.: 617-373-2385; Fax:
617-373-3724; E-mail:
spl{at}neu.edu.
1 The abbreviations used are: CPS, carbamoyl phosphate synthetase; AGA,
N-acetylglutamate; CP, carbamoyl phosphate; fCPS, frog CPS; eCPS,
E. coli CPS; GAT, glutamine amidotransferase; SYN, synthetase. 
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ACKNOWLEDGMENTS
|
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We thank Quinn Lu for the plasmid pESP-5 and Michael Kothe for a critical
review of this manuscript.
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