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INTRODUCTION |
During the synthesis of various biomolecules including amino
acids, nucleotides, and coenzymes, the amido group of glutamine is
transferred to a large variety of acceptor substrates by glutamine amidotransferases (GATases)1
(1, 2). GATases catalyze two separate reactions at two active sites
that are either located on a single polypeptide chain or on different
subunits. In the glutaminase reaction, glutamine is hydrolyzed to
glutamate and ammonia, which in the synthase reaction is added to an
acceptor substrate that is specific for each GATase.
There are two classes of GATases that can be discriminated by
catalytically essential residues in their glutaminase domains (1, 3).
The key feature of class I GATases is the catalytic triad Cys-His-Glu.
Recent x-ray structure determinations of three class I GATases,
Escherichia coli carbamoyl phosphate synthase (4, 5),
E. coli GMP synthase (6), and S. solfataricus anthranilate synthase (7) indicate a common fold of their glutaminase domains, which is similar to the well known
/
hydrolase fold (8).
Class II GATases belong to the large family of Ntn hydrolases (9), and
their only catalytically essential amino acid is the conserved
N-terminal cysteine. The corresponding synthase domains within each
class are structurally, evolutionary, and functionally unrelated (2),
supporting the hypothesis that glutamine-hydrolyzing enzymes were
recruited independently by previously ammonium-dependent enzymes (3). Along these lines, most GATases can use ammonium salts as
an alternative source of ammonia (1, 2).
The imidazole glycerol phosphate (ImGP) synthase is a class I GATase,
which in bacteria constitutes a bienzyme complex consisting of the
glutaminase subunit HisH and the synthase subunit HisF (10, 11). The
ammonia produced by HisH reacts with the substrate of HisF, which is
N'-((5'-phosphoribulosyl)
formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR). The products of this reaction, ImGP and
5- aminoimidazole-4-carboxamide ribotide (AICAR), are further used in
histidine and de novo purine biosynthesis, respectively. In
yeast, the glutaminase and synthase activities are located on a single
polypeptide chain, which is termed HIS7 (12, 13). The mechanism
of glutamine hydrolysis by HisH can be deduced from the class I GATase
carbamoyl phosphate synthase (5). However, due to the lack of a high
resolution structure for the bienzyme complex, the ammonia
transfer from HisH to HisF and the mechanism of the HisF reaction are
only poorly understood.
In order to address these questions, the thermostable variants tHisF
and tHisH from Thermotoga maritima were produced in E. coli, purified and characterized by hydrodynamic and spectroscopic measurements, limited proteolysis, and steady-state enzyme kinetics. Moreover, the high resolution x-ray structure of isolated tHisF (14)
was used to identify and probe amino acid residues that are potentially
involved in catalysis of the synthase reaction. It was shown that tHisH
is activated by complex formation with tHisF containing ImGP or a
substrate analogue bound to its active site. A flexible loop region in
tHisF appears to be important for these functional interactions with
tHisH. Furthermore, two aspartate residues at the active site of tHisF
were demonstrated to be essential for catalysis. One of them was
partially replaceable by glutamate, as shown by saturation random
mutagenesis and complementation in vivo of an E. coli
hisF deletion strain. Based on these findings, a chemically
plausible mechanism for the HisF synthase reaction was derived, which
involves general acid/base catalysis.
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EXPERIMENTAL PROCEDURES |
DNA Manipulation and Sequence Analysis--
Preparation of DNA,
digestion with restriction endonucleases, and DNA ligation were
performed as described (15). Oligonucleotides were purchased from
Metabion. DNA was amplified by PCR using cloned Pfu
polymerase (Stratagene). For PCR with mutagenic oligonucleotides, Taq polymerase (Roche Diagnostics) was used. DNA was
extracted from agarose gels using the QIAquick gel extraction kit
(Qiagen). DNA sequencing was performed by the "Göttingen
Genomics Laboratory" and by the "Zentrum für Molekulare
Medizin der Universität Köln," using standard methods.
N-terminal protein sequencing was performed by Dr. Paul Jenö
(Biozentrum, University of Basel) and the "Zentrum für
Molekulare Medizin der Universität Köln," again using
standard methods.
Subcloning of the hisF and hisH Genes from T. maritima--
The
hisF gene variants from Thermotoga maritima
(thisF; Ref. 16) encoding the synthase subunit of imidazole
glycerol phosphate synthase were cloned into the expression vector
pET11c (Novagen) using the restriction enzymes NdeI and
BamHI. The hisH gene of T. maritima
(thisH; Ref. 16) was amplified by PCR, using purified chromosomal DNA (Qbiogen) as a template. The oligonucleotides 5'-GGT GTG ATA GCA TGC GTA TCG-3' with a
SphI-site (in boldface type) and 5'-CTA CCA AGC
TTC TGA AGA GAT CTA TCG-3' with a HindIII-site (in
boldface type) were used as 5'- and 3'-primers, respectively. Using the
two newly introduced restriction sites, the amplified DNA fragment was
cloned into the vector pDS56/RBSII/SphI (17) to yield the
plasmid pDS56/RBSIISphI-thisH. All inserts were
entirely sequenced to exclude inadvertent PCR mutations.
Site-directed Mutagenesis of thisF--
Point mutations were
introduced into thisF by PCR-based methods using either
mutagenic 5'-primer for construction of thisF_C9A, D11N,
K19S, or the megaprimer method (18) for construction of thisF_D51N, N103A, D130N, D176N, and D183N. In both
approaches, the plasmid pET11c-thisF (19) was used as the
template. The following mutagenic 5'-primers were used (NdeI
restriction sites are in boldface type, and base substitutions to
introduce an amino acid exchange are underlined): 5'-TGA TGA AGA
CAT ATG CTC GCT AAA AGA ATA ATC GCG GCT CTC
GAT-3' for construction of C9A, 5'-TGA TGA AGA CAT ATG CTC
GCT AAA AGA ATA ATC GCG TGC CTC AAT GTG AAA GAC-3' for
construction of D11N, and 5'-TGA TGA AGA CAT ATG CTC GCT AAA
AGA ATA ATC GCG TGT CTC GAT GTG AAA GAC GGT CGT GTG GTG AGC
GGA ACG AAC TTC-3' for construction of K19S. In all PCRs, the
oligonucleotide 5'-CCG GAT CCA GCG TCA TCA CAA-3' containing
a BamHI restriction site (in boldface type) was used as the
3'-primer. For the production of megaprimers, the following mutagenic
oligonucleotides were used (base substitutions to introduce restriction
sites for the control of the reaction are in boldface type, and base
substitutions to introduce an amino acid exchange are underlined):
5'-CGC GGT AAT ATT CAG AAA AAC
GAG-3' with a new SspI restriction site for construction of D51N, 5'-CAC AGC CGC AGT GGC TAT GCT
CAC CTT GTC-3' with a new TspRI site for construction of
N103A, 5'-CAC TCT TTT TGC ATT
AAT CGC CAC GAC-3' with a new
VspI restriction site for construction of D130N, 5'-GAC AGA
AAC GGC ACC AAA TCG G-3' with a new
BshNI restriction site for construction of D176N, and 5'-GGC
ACA AAA TCG GGT TAC AAC
ACT GAG ATG ATA
AGG-3' with a new TspRI restriction site for construction of
D183N. For production of the megaprimers for thisF_D51N,
N103A, and D130N, the corresponding mutagenic oligonucleotides listed
above were used as 3'-primers, and the oligonucleotide 5'-TGA TGA AGA
CAT ATG CTC GCT AAA AG-3' (NdeI restriction site
in boldface type) was used as 5'-primer. The megaprimers were purified
by agarose gel electrophoresis and used in the second PCR as
5'-primers, whereas the oligonucleotide 5'-CCG GAT CCA GCG
TCA TCA CAA-3' (BamHI restriction site in boldface type) was
used as 3'-primer. The construction of the variants
thisF_D176N and D183N followed the same protocol except that
in the first PCR the mutagenic oligonucleotides were used as 5'-primers
and the oligonucleotide 5'-CCG GAT CCA GCG TCA TCA CAA-3'
(BamHI restriction site in boldface type) was used as
3'-primer. The purified megaprimers were used as 3'-primers, and the
oligonucleotide 5'-TGA TGA AGA CAT ATG CTC GCT AAA AG-3'
(NdeI restriction site in boldface type) was used as
5'-primer. The resulting full-length products were digested with
NdeI and BamHI and ligated into pET11c. In order
to confirm the base substitutions and to exclude inadvertent additional
ones, all thisF gene variants were entirely sequenced.
Randomization of thisF Codons--
The thisF codons
representing amino acids 11 and 130 were randomized in PCR-based
approaches using degenerated primers and pET11c-thisF as
template. For randomization of position 11, the oligonucleotide 5'-TAT
ACG CAT GCT CGC TAA AAG AAT AAT CGC GTG CCT CNN
SGT GAA GAC-3' with a SphI restriction site (in boldface type) was used, where N represents equal molar
mixtures of all four bases, and S represents an equal molar
mixture of G and C. In a PCR, the degenerate oligonucleotide was used
as 5'-primer, and the oligonucleotide 5'-GTC GAC GGA TCC ACA ACC CCT CCA G-3' with a BamHI restriction site (in boldface
type) was used as 3'-primer to yield an ensemble of
thisF_D11NNS gene variants. For randomization of position
130, a megaprimer was produced using the degenerate oligonucleotide
5'-GTC GTG GCG ATT NNS GCA AAA AGA-3' as 3'-primer and the
oligonucleotide 5'-TAT ACG CAT GCT CGC TAA AAG AAT AAT
CGC-3' with a SphI restriction site (in boldface type) as
5'-primer. In a second PCR, the purified megaprimer was used as
5'-primer, and the oligonucleotide 5'-GTC GAC GGA TCC ACA
ACC CCT CCA G-3' with a BamHI restriction site (in boldface
type) was used as 3'-primer to yield an ensemble of
thisF_D130NNS gene variants.
In Vivo Complementation--
The PCR-amplified ensembles of
thisF genes containing randomized codons at amino acid
position 11 or 130 were ligated into a modified pDS56/RBSII vector
(termed pTNA), which contains a truncated derivative of the
tryptophanase operon promotor (20) that permits constitutive gene
expression in E. coli. The auxotrophic E. coli
strain UTH860 (
hisF), which carries a mutant
ehisF gene that encodes an inactive HisF protein (21), was
transformed separately with the two plasmid libraries and plated onto
LB medium containing 150 µg ml
1 ampicillin
(15). The resulting lawns of at least 105 clones were
rinsed off the plates, and plasmid DNA was prepared from the mixture of
colonies. The
hisF strain was retransformed with these
plasmid libraries, and aliquots were streaked on LB medium plates
(nonselective plates) or on minimal medium plates (22) without
histidine (selective plates) and incubated at 37 °C. From the
nonselective plates, 16 colonies with randomized thisF codon
11 and 18 colonies with randomized thisF codon 130 were
picked, and the corresponding thisF genes were entirely sequenced.
The mutational saturation is given by the following,
|
(Eq. 1)
|
where p is the probability that, for
m randomized codons appearing with the relative frequency
fi, practically all possible amino acid combinations
are present in a library containing n independent clones
(23). Under the assumption that all 32 permitted codons appear with
equal frequency (fi =
), mutational
saturation (p
0.99) of one codon (m = 1) is attained with a library of more than 145 independent clones.
Although the bases within codons 11 and 130 were not randomly
distributed, every allowed base was found at all three codon positions.
None of the thisF genes contained additional mutations.
Considering the size of the two libraries, each of which contained at
least 105 independent clones, it can be concluded that all
20 amino acids were represented at both randomized positions. To select
for functional amino acids at position 11 or 130 of tHisF, for each
library one selective plate was incubated for various time periods. A
number of clones appeared on both plates overnight and, after 48 h, additional colonies appeared on the selective plate with
thisF genes that were randomized at position 130. Incubation
was continued for 1 week, but no additional colonies appeared. A number
of colonies that grew on selective medium after different periods of
time were picked, and the thisF_D11NNS or
thisF_D130NNS genes that encoded functional tHisF
proteins were sequenced.
Purification of tHisF, tHisF Variants, and tHisH--
Wild-type
tHisF and its variants containing individual amino acid exchanges were
purified as described (19). The yield was between 7 and 17 mg of
purified enzyme per g of wet cell mass. Heterologous expression of
thisH was conducted in E. coli W3110
trpEA2
cells containing pDS56/ RBSIISphI-thisH and the
repressor plasmid pDMI, 1, as described for hisA from
T. maritima (19). The cells were grown in 1 liter of LB
medium supplemented with 0.15 mg/ml ampicillin and 0.075 mg/ml
kanamycin. Overexpression of thisH was induced by adding 1 mM isopropyl-1-thio-
-D-galactopyranoside at
an optical density at 600 nm of about 0.6, and incubation was continued
overnight. The cell suspension was washed with 100 mM potassium phosphate buffer at pH 7.5, containing 1 mM EDTA
and 1 mM dithiothreitol, resuspended (5 ml of buffer per g
wet cell mass), and lysed by sonification (Branson Sonifier W-250,
2 × 2 min, 50% pulse, 0 °C). According to SDS-PAGE, about
60% of tHisH were found in the soluble fraction of the cell extract.
Benzonase (Merck) (50 units) was added to this fraction, which was then incubated for 1 h at 37 °C and subsequently for 20 min at
75 °C. The resulting suspension was centrifuged (Sorvall SS34,
12,000 rpm, 30 min, 4 °C), and the pellet, which contained
heat-labile host proteins, was discarded. The supernatant was dialyzed
against 10 mM Tris/HCl buffer at pH 8.0, containing 1 mM EDTA and 1 mM dithiothreitol, and loaded on
an anion exchange column (POROS HQ20; 1 × 10 cm, PE Biosystems)
that was equilibrated with the same buffer at room temperature. The
column was washed with four volumes of equilibration buffer, and bound
proteins were eluted with 1.5 liters of a linear gradient of 0-1
M sodium chloride at pH 8.0. tHisH eluted between 130 and
150 mM sodium chloride, as judged from SDS-PAGE and
conductivity measurements. Fractions containing tHisH were pooled,
dialyzed against 10 mM potassium phosphate buffer at pH
7.5, containing 1 mM dithiothreitol, loaded on a
hydroxylapatite column (3.6 × 20 cm; Novartis) that was
equilibrated with the same buffer, and eluted with 2 liters of a linear
gradient of 10-500 mM potassium phosphate. tHisH eluted
between 100 and 150 mM potassium phosphate with a purity
above 95%, as judged from SDS-PAGE. The purification yielded ~5 mg
of tHisH per g of wet cells. Fractions containing pure tHisH were
pooled, dialyzed against 50 mM potassium phosphate buffer
at pH 7.5, containing 1 mM EDTA and 1 mM
dithiothreitol, concentrated to 1.8 mg/ml using Centricon-10
concentration devices (Millipore), and shock-frozen in liquid nitrogen.
Analytical Methods--
Purification of proteins was followed by
electrophoresis on 12.5 or 15% SDS-polyacrylamide gels using the
system of Laemmli (24) and staining with Coomassie Blue. During
purification, the protein concentration was determined according to
Bradford (25). The concentration of purified proteins was determined with molar extinction coefficients at 280 nm that were calculated from
the amino acid sequence (26). Analytical gel filtration was performed
at a flow rate of 0.5 ml/min on a Superdex 75 column (1 × 30 cm;
Amersham Pharmacia Biotech) that was equilibrated with 50 mM potassium phosphate at pH 7.5, containing 300 mM sodium chloride. Apparent molecular masses were
determined from the corresponding elution volumes, using a
calibration curve that was obtained with standard proteins.
Sedimentation equilibrium runs were performed in a Beckman analytical
ultracentrifuge (model Optima XLA), following the absorption at 278 nm.
Runs with tHisF were performed as described (27). Runs with tHisH were
performed at 24,000 rpm and protein concentrations of 12 and 23 µM in 50 mM potassium phosphate, pH 7.5, at
20 °C, containing 25 mM potassium chloride. Runs with tHisH-tHisF were performed at 18,000 and 24,000 rpm with 20 µM protein in 50 mM potassium phosphate at pH
7.5 at 20 °C, containing 300 mM sodium chloride. To
determine apparent molecular masses, the runs were analyzed as
described (27). Fluorescence spectra were recorded with a F-4500
spectrofluorimeter (Hitachi) or a Cary Eclipse spectrofluorimeter
(Varian). Proteolytic stability was tested at room temperature by
incubating 10 nmol of substrate protein with 64 pmol of trypsin in 1 ml
of 50 mM potassium phosphate, pH 7.5. The reaction was
stopped after different time intervals by adding one volume of 2×
SDS-PAGE sample buffer and heating for 5 min at 95 °C. The time
course of proteolysis was followed on Tris-Tricine gels containing 20%
acrylamide (28).
Steady-state Enzyme Kinetics--
The
ammonia-dependent activity of isolated tHisF was measured
by recording entire progress curves in 50 mM Tris acetate
buffer, pH 8.5, at 25 °C. In order to determine
KmPRFAR, the enzyme was saturated with
ammonia by adding 100 mM ammonium acetate corresponding to
14.4 mM NH3 at pH 8.5. PRFAR, 20 µM, were synthesized in situ from ProFAR,
using a molar excess of HisA from T. maritima (19) and
completely converted by tHisF to ImGP and AICAR. The reaction was
quantified by the decrease in absorption at 300 nm, using

300(PRFAR-AICAR) = 5.64 mM
1 cm
1
(11). In order to determine
KmNH3,
the reaction was performed in the presence of 50 µM PRFAR
at various concentrations of ammonium acetate between 0 and 200 mM, corresponding to 0 and 35 mM
NH3 at pH 8.5. The glutamine-dependent activity
of the tHisH-tHisF complex was measured in an analogous way as the
ammonia-dependent reaction of tHisF, but the pH value was
set to 8.0. In order to determine
KmPRFAR, the reaction was performed with
20 µM ProFAR and 5 mM
L-glutamine. To determine
KmGln, the reaction was performed in the
presence of 50 µM PRFAR at various concentrations of
glutamine between 0 and 7 mM. The progress curves were
analyzed with the integrated form of the Michaelis-Menten equation
(29), yielding values for Km and
Vmax. The glutaminase activity of tHisH in
complex with liganded tHisF was measured in a coupled enzymatic assay
with bovine liver glutamate dehydrogenase (Sigma). Glutamate that was
produced by tHisH was oxidized by a molar excess of glutamate
dehydrogenase in the presence of NAD+, yielding
2-oxoglutarate and NADH + H+ + NH4+. The reaction was quantified by the
increase in absorption at 340 nm, using

340(NADH-NAD+) = 6300 M
1 cm
1.
The values for KmGln and
Vmax were deduced from initial velocity
measurement at various glutamine concentrations.
 |
RESULTS |
Production and Purification of tHisF, tHisH, and the tHisH-tHisF
Complex--
tHisF was produced and purified as described previously
(19). The thisH gene (16) was cloned into the expression
vector pDS56/RBSII/SphI. Proteins were expressed in E. coli from this plasmid under the control of a lac
operator system (17). tHisH was purified from the soluble fraction of
the cell homogenate by first heat-precipitating E. coli host
proteins, followed by anion exchange and hydroxylapatite
chromatography. The final preparation was more than 98% pure as judged
by SDS-PAGE and gel filtration chromatography on Superdex 75 (data not
shown). The presence of the N-terminal methionine residues of both
purified tHisF and tHisH were confirmed by N-terminal protein
sequencing. The complex of tHisF and tHisH (tHisH-tHisF) was prepared
by mixing equal molar amounts of both proteins, followed by gel
filtration chromatography in order to remove any unintentional surplus
of either of the components. The concentration of the purified proteins
was determined by absorption spectroscopy, using calculated molar
extinction coefficients at 280 nm (26) of 11,500 M
1 cm
1
for tHisF, 17,400 M
1
cm
1 for tHisH, and 28,900 M
1 cm
1
for the tHisH-tHisF complex.
Association States--
The molecular masses and the association
states of recombinant tHisF, tHisH, and tHisH-tHisF were determined by
analytical gel filtration chromatography and equilibrium analytical
ultracentrifugation. tHisF eluted from a calibrated Superdex 75 column
with an apparent molecular mass of 26.4 kDa (27), which is similar to
the molecular mass for the monomer as calculated from the amino acid
sequence (27.7 kDa). tHisH eluted with a significantly lower apparent
molecular mass (17.4 kDa) than the calculated molecular mass for the
monomer (23.1 kDa). A similarly retarded elution has been observed
previously for eHisH (11), for unknown reasons. Mixed equal molar
amounts of tHisH and tHisF eluted as a single peak with an apparent
molecular mass of 41.8 kDa, which is smaller than the calculated
molecular mass for the 1:1 complex (50.8 kDa). In order to further
clarify their association states, the molecular masses of tHisH, tHisF, and tHisH-tHisF were determined by analytical ultracentrifugation. The
data from several independent sedimentation equilibrium runs yielded
average molecular masses of 24.6 kDa for tHisH, 49.8 kDa for
tHisH-tHisF, and, dependent on protein concentration, between 26.0 and
38.4 kDa for tHisF (27). These results confirm that isolated tHisH and
tHisF are essentially monomeric proteins that assemble to a stable 1:1
heterodimeric bienzyme complex. These association states of the
T. maritima proteins are identical to those of the E. coli homologs (11), which excludes the idea that a higher
association state is responsible for the increased thermostability of
the ImGP synthase from the hyperthermophilic compared with that of the
mesophilic bacterium (30).
Fluorescence Spectroscopic Analysis--
Fluorescence emission
spectra were monitored to determine the relative solvent accessibility
of the single tryptophan residues in tHisF (Trp156) and
tHisH (Trp123). As judged from the emission maxima at 323 and 339 nm, Trp156 in tHisF appears to be shielded from
solvent, while Trp123 in tHisH appears to be partly exposed
to solvent (Fig. 1a). A solution containing equal molar amounts of tHisF and tHisH showed an
emission maximum at 326 nm, indicating that complex formation leads to
the burial of Trp123 of tHisH but does not change the
solvent accessibility of Trp156 in tHisF. Along these
lines, the titration of tHisH with tHisF leads to a linear shift of the
emission maximum from 339 nm to lower wavelengths, until approximately
equal molar amounts of tHisH and tHisF are present. An excess of tHisF
does not lead to a further shift of the emission maximum (Fig.
1b). These data suggest that, at the applied protein
concentrations, the binding of tHisF and tHisH is stoichiometric,
indicating a high affinity of the two proteins. Although the
thermodynamic dissociation constant Kd cannot be
determined by this method, it has to be much smaller than the
equivalence concentration, which is 10 µM.

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Fig. 1.
Fluorimetric analysis of tHisF,
tHisH, and the tHisH-tHisF complex. Conditions were as follows:
excitation wavelength, 295 nm; 10 µM enzyme in 10 mM potassium phosphate buffer, pH 7.5, at 25 °C.
a, different solvent accessibility of tryptophan residues.
The single tryptophan residue of tHisF has its emission maximum at 323 nm, indicating that it is shielded from solvent. The single tryptophan
residue of tHisH has its emission maximum at 339 nm, indicating that it
is partly accessible to solvent. In the tHisH-tHisF complex, the
emission maximum lies at 326 nm, indicating that both tryptophan
residues are shielded from solvent. b, formation of the
stoichiometric tHisH-tHisF complex. 10 µM tHisH was
titrated with tHisF, and complex formation was followed by a shift of
the fluorescence emission maximum (Femmax)
from 339 nm to lower wavelengths. The titration curve is linear up to
about equal molar concentrations of tHisF and tHisH and remains
constant at higher ratios, indicating that the thermodynamic
dissociation constant for complex formation, Kd, is
10 µM.
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Steady-state Enzyme Kinetics--
The catalytic activities of
isolated tHisF and tHisH and of the tHisH-tHisF complex were measured
under steady-state conditions with coupled enzymatic assays, using
absorption spectroscopy (Fig. 2). The
synthase activities of the tHisH-tHisF complex and of isolated tHisF
were measured, using glutamine and ammonium acetate as ammonia donors,
respectively (Fig. 2, b and c). The glutaminase activity of tHisH was measured in the presence and absence of tHisF and
the ligands ImGP or ProFAR (Fig. 2d), which were shown to
activate the glutaminase activity of eHisH when bound to the active
site of eHisF (11). Entire progress curves or initial velocities were
measured and fitted to the integrated or the simple Michaelis-Menten
equation, respectively. The resulting kcat and Km values were compared with those of the E. coli homologs eHisH, eHisF, and eHisH-eHisF (11) and with those of
HIS7 from Saccharomyces cerevisiae, in which the glutaminase
and the synthase domains are fused (13).

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Fig. 2.
The reactions catalyzed by the tHisH-tHisF
complex and the individual enzymes were monitored spectroscopically
using coupled enzymatic assays. a, physiological
reaction of the imidazole glycerol phosphate synthase (tHisH-tHisF).
b, tHisH-tHisF assay. PRFAR was synthesized in
situ from ProFAR with a molar excess of HisA from T. maritima (tHisA); the glutamine-dependent
conversion of PRFAR into ImGP and AICAR was quantified using
 300(PRFAR-AICAR) = 5640 M 1 cm 1 (11). c,
tHisF assay. The ammonia-dependent reaction was measured by
replacing glutamine with ammonium acetate. d, tHisH assay.
The glutamate produced by the glutaminase activity of tHisH was
oxidized by a molar excess of glutamate dehydrogenase (GDH)
to 2-oxoglutarate and ammonium; the reaction was quantified by the
concomitant reduction of NAD+ to NADH + H+,
using  340(NADH-NAD+) = 6300 M 1 cm 1.
The ligand L bound to tHisF is either ImGP or ProFAR. The conditions
for the different assays are listed in Table I (for the synthase
reactions) and Table II (for the glutaminase reaction).
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|
Table I summarizes the results of
the synthase activity measurements. In the
ammonia-dependent reaction, at comparable temperatures and
pH values, isolated tHisF exhibited 2-5-fold higher catalytic efficiencies
(kcat/KmPRFAR and
kcat/KmNH3)
than isolated eHisF. The higher catalytic efficiency was caused by
lower Km values of tHisF for both PRFAR and ammonia, while the turnover rate kcat was slightly lower
for tHisF than for eHisF. In the glutamine-dependent
reaction, the tHisH-tHisF complex exhibited catalytic activities
comparable to tHisF in the ammonia-dependent reaction. In
contrast, the catalytic efficiency kcat/KmPRFAR of
the eHisH-eHisF complex in the glutamine-dependent reaction was about 25-fold higher than that of eHisF in the
ammonia-dependent reaction, due to a much higher
KmPRFAR value of eHisF. The decreased
activity of eHisF in the ammonia-dependent reaction might
be due to inhibition by high concentrations of ammonium chloride, the
ammonia donor (11). tHisF was also inhibited by ammonium chloride,
which was therefore replaced by ammonium acetate that showed no
detectable inhibitory effect at the applied concentrations (data not
shown). Similar catalytic efficiencies were observed for the
glutamine-dependent synthase reactions of the bifunctional
HIS7 enzyme and the tHisH-tHisF complex (Table I).
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Table I
Steady-state kinetic constants of the ammonia-dependent
ImGP synthase reaction of isolated HisF subunits and the
glutamine-dependent synthase reaction of HisH-HisF
complexes
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In the absence of tHisF, tHisH did not show any measurable glutaminase
activity. However, as observed with the E. coli enzyme (11),
the binding of either the product ImGP or the substrate analogue ProFAR
to the active site of tHisF led to a strong stimulation of tHisH. In
contrast, neither the T. maritima, nor the E. coli HisH glutaminase activities are stimulated by AICAR, the
second product of the ImGP synthase reaction (Ref. 11 and data not shown). The steady-state parameters of the glutaminase activity of
tHisH were therefore measured in the presence of tHisF and saturating
concentrations of either ImGP or ProFAR (Table
II). The kcat of
the glutaminase reaction is 0.1 s
1 and lower
by a factor of about 4-8 than the kcat value of
the glutamine-dependent synthase reaction (Table I), where
PRFAR (instead of ImGP or ProFAR) is bound to the active site of tHisF in the tHisH-tHisF complex. Similarly, in the E. coli
enzyme, the kcat of the glutaminase reaction is
lower by a factor of 3-4 than the kcat of the
glutamine-dependent synthase reaction. In addition, the
Km values for glutamine are increased about 10-fold
(Tables I and II). Obviously, both in the T. maritima and
the E. coli ImGP synthase, the activation of the glutaminase reaction by the synthase subunit is more efficient when the native substrate PRFAR is bound rather than ImGP or ProFAR. In contrast, in
HIS7, where glutaminase and synthase activities are located on the same
bifunctional polypeptide chain, ImGP and ProFAR are equally efficient
glutaminase activators as PRFAR (Tables I and II). The dependence of
the glutaminase activity of tHisH on the presence of a ligand bound to
the active site of tHisF can be used to estimate the thermodynamic
dissociation constants Kd for the binding of these
ligands. As shown in Table II, ImGP binds with similarly low affinity
to tHisF and eHisF, whereas ProFAR binds almost 20-fold more
strongly to tHisF than to eHisF.
Limited Proteolysis--
The sensitivity of a protein toward
proteolytic degradation can provide valuable information on its
stability as well as on the flexibility of potential cleavage sites
(31). For this reason, tHisF, tHisH and tHisH-tHisF were digested with
trypsin, and the results were analyzed by SDS-PAGE (Fig.
3). Isolated tHisF (27.7 kDa) was cleaved
once to yield fragments with apparent molecular masses of about 24 kDa
(Fig. 3a) and about 4 kDa (not shown). These fragments,
which were detectable already after 5 min of incubation, were enriched
at the cost of intact tHisF in a time-dependent manner.
After 120 min, about one-quarter of tHisF was degraded (Fig.
3a). N-terminal sequencing of the large fragment showed that
cleavage occurred C-terminal of Arg27 (Fig.
4). Arg27 is located in a
long and presumably flexible region at the C-terminal (active site)
face of the
-barrel (cf. arrow in Fig.
4a). In contrast to tHisF, at the given conditions isolated
tHisH was completely resistant to trypsin (data not shown). Incubation
of the tHisH-tHisF complex with trypsin again resulted in the
degradation of tHisF, albeit with significantly higher rate; after 120 min, almost all tHisF was degraded into fragments of about 24 kDa (Fig. 3b) and about 4 kDa (not shown). These results show that the
single trypsin cleavage site at Arg27 is more susceptible
in complexed tHisF compared with isolated tHisF.

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Fig. 3.
Isolated tHisF is less susceptible to
proteolytic attack than complexed tHisF. 10 µM
isolated tHisF (a) or a 10 µM concentration of
the tHisH-tHisF complex (b) was digested with 64 nM trypsin for the indicated time intervals in 50 mM potassium phosphate, pH 7.5, at 25 °C. tHisF (27.7 kDa) is degraded to one large fragment with Mapp
of ~24 kDa and one small fragment with Mapp of
~4 kDa (not shown). This cleavage, which takes place C-terminal of
Arg27 (cf. arrow in Fig. 4), occurs more slowly
in isolated compared with complexed tHisF, indicating conformational
changes in the flexible loop region between strand 1 and
helix 1. tHisH (23.1 kDa) is resistant to trypsin, both
in the tHisH-tHisF complex (b) and in the isolated form (not
shown). Band 1, tHisF; band
2, large tryptic fragment of tHisF (a) or a
mixture of tHisH and the large tryptic fragment of tHisF
(b). M, standard proteins.
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Fig. 4.
Conserved and catalytically important amino
acid residues in the
( )8-barrel structure
of tHisF (14). a, structure-based sequence alignment.
Secondary structural elements are identified by blue
arrows ( strands) and red cylinders
( helices). Conserved, amino acids that are invariant in
all known 25 HisF sequences and identical in at least 22 sequences are
shown in uppercase and lowercase, respectively.
Exchanged, eight conserved amino acid residues close to the
proposed active site of tHisF were replaced individually with
structurally similar residues (in boldface type).
Amino acids involved in phosphate binding are underlined.
The single trypsin cleavage site within tHisF, which is located
C-terminal to Arg27, is marked by an arrow
(cf. Fig. 3). b, ribbon diagrams showing a side
view on the central -barrel. P(N) and
P(C) are the phosphate ions bound to the N- and
C-terminal halves of tHisF. Asp11 and Asp130
(D11 and D130) are essential for catalysis of the
tHisF reaction, Asp176 (D176) appears to be
important for catalysis, and the flexible region containing
Lys19 (K19) and Arg27
(R27) is involved in interactions between tHisF and tHisH
that take place upon the reaction of nascent ammonia with PRFAR (side
chains in green). Side chains of conserved amino acids at
the N-terminal face of the -barrel, which might be involved in
binding of tHisH, are in black (see "Results" and
"Discussion" for details.)
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Mutational Analysis of the Active Site of tHisF--
Glutamine
hydrolysis reactions catalyzed by other members of the class I GATase
family have been characterized in detail, for example in the case of
carbamoyl phosphate synthase (5, 32, 33). In contrast, the complicated
reaction mechanism that leads to the formation of the imidazole ring
catalyzed by HisF (Fig. 2a) has not been investigated so
far. The first step toward this goal is to identify the catalytic amino
acid residues. These should (a) be invariant in the 25 currently available HisF sequences; (b) lie at the
C-terminal face of the central
-barrel of tHisF, which is the
location of the active sites of all known (
)8-barrel enzymes (34); and (c) be able to reversibly provide and abstract a
proton, since the HisF reaction probably involves general acid/base catalysis. Eight residues that fulfill these criteria were replaced by
site-directed mutagenesis with structurally similar amino acids lacking
the putative functional group (Fig. 4a). The mutant
thisF genes were expressed in E. coli, and the
respective gene products were purified in the same way as described for
the wild-type protein (19). In order to exclude structural
perturbations (or denaturation) caused by the introduced amino acid
replacements, all purified tHisF variants were analyzed by fluorescence
spectroscopy and analytical gel filtration chromatography. No
significant difference from wild-type tHisF was detected in any
of the variants, confirming the assumption that residues at the
C-terminal face of (
)8-barrels do not contribute
significantly to protein stability (35). Also, the association with
tHisH was not impaired (data not shown).
The steady-state enzyme kinetic constants of the isolated tHisF
variants and of the corresponding tHisH-tHisF complexes were determined
and compared with the constants of the wild-type enzymes (Tables
III and
IV). The catalytic efficiencies
kcat/Km of both isolated and
complexed tHisF_C9A, tHisF_D51N, tHisF_N103A, and tHisF_D183N were not
significantly different from wild-type tHisF, ruling out any central
catalytic role for the replaced residues. Also, the
ammonia-dependent reactions of isolated tHisF_K19S were
similarly efficient as those of wild-type tHisF (Table III). In
contrast, the efficiencies of the glutamine-dependent
reactions of the tHisH-tHisF_K19S complex were significantly impaired
(Table IV). The variant tHisF_D176N showed a 40-50 fold decrease in
kcat, both in isolated form and in complex with
tHisH. The strongest effects, however, were found for tHisF_D11N and
tHisF_D130N, the catalytic efficiencies
kcat/KmPRFAR of
which were decreased by approximately 5 orders of magnitude. While for
tHisF_D11N only the kcat was affected, both
kcat and Km were drastically
impaired in tHisF_D130N. These results suggest that Asp11
and Asp130 play essential roles in the catalysis of the
tHisF reaction.
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Table III
Effect of individual amino acid exchanges on the steady-state enzyme
kinetic constants of the ammonia-dependent ImGP synthase
reaction catalyzed by isolated tHisF
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Table IV
Effect of individual amino acid exchanges on the steady-state enzyme
kinetic parameters of the glutamine-dependent ImGP synthase
reaction catalyzed by the tHisH-tHisF complex
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Saturation Random Mutagenesis and Complementation in Vivo--
To
further test the crucial role of Asp11 and
Asp130 for catalysis, both residues were replaced by all 19 alternative amino acids, and their function in catalysis was probed by
selection in vivo. To this end, the corresponding codons at
amino acid positions 11 or 130 were subjected to saturation random
mutagenesis using degenerate oligonucleotides. The randomization of the
third codon position was limited to G and C. This restriction
eliminates 32 out of 64 codons, but the remainder codons still
represent all 20 amino acids.
Histidine auxotrophic E. coli cells lacking a functional
hisF gene (
hisF cells) were transformed with a
plasmid that allows constitutive expression in E. coli of
the cloned thisF_D11NNS or thisF_D130NNS
ensembles (20). Transformants were streaked onto selective medium
without histidine and incubated at 37 °C; a small aliquot was
streaked onto nonselective LB medium. From the nonselective plates,
colonies containing thisF_D11NNS or thisF_D130NNS were randomly picked, and the thisF genes were sequenced in
order to confirm the random distribution of bases at each position of the codons. Sequencing of thisF from colonies grown on
selective medium showed that at position 11 only the wild-type amino
acid aspartate allowed functional complementation of the
hisF cells. Similarly, all overnight grown colonies on
the selective plate with the randomized codon at position 130 coded for
the wild-type amino acid aspartate. However, all colonies that appeared
on this plate after 48 h contained a codon for glutamate at
position 130 (Table V).
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Table V
Asp11 is essential for tHisF function, but Asp130 can
be functionally replaced by Glu
Results of saturation mutagenesis and functional complementation
in vivo are shown.
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In order to test its catalytic activity in vitro,
tHisF_D130E was produced in E. coli, purified and
characterized as described above for the other tHisF variants.
tHisF_D130E was identical to the wild-type enzyme with respect to
fluorescence properties and complex formation with tHisH (data not
shown). Its steady-state enzyme kinetic parameters, however, were
between those of wild-type tHisF and tHisF_D130N; the
kcat value was reduced by a factor of about
400-500, and the KmPRFAR was
increased almost 20-fold (Table III).
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DISCUSSION |
Catalytic Properties and Allosteric Interactions of T. maritima
ImGP Synthase--
Most investigated enzymes from the
hyperthermophilic bacterium T. maritima are only marginally
active at room temperature, probably due to conformational rigidity at
their active sites (36). In contrast, at comparable temperatures of 25 and 30 °C, the isolated synthase subunit tHisF shows a higher
catalytic efficiency (kcat/KmPRFAR and
kcat/KmNH3)
than eHisF, due to much lower Km values (Table I). Similarly, both phosphoribosyl anthranilate isomerase and
indoleglycerol phosphate synthase from T. maritima, which
are involved in tryptophan biosynthesis, have lower
Km values and higher catalytic efficiencies than
their homologues from E. coli (37, 38). It has been
speculated that the high catalytic efficiency of T. maritima
phosphoribosyl anthranilate isomerase is important for rapid processing
of its thermolabile substrate, and a similar argument may hold for the
procession by tHisF of the extremely labile PRFAR (39). However, the
catalytic efficiencies of the overall tHisH-tHisF reaction
(kcat/KmPRFAR and
kcat/KmGln; Table
I) and of the glutaminase reaction
(kcat/KmGln;
Table II) were significantly lower than those of the corresponding enzymes from E. coli and S. cerevisiae. Thus, the
catalytic activity of tHisH seems to limit, at least at room
temperature, the overall catalytic efficiency of T. maritima
ImGP synthase. This result indicates conformational rigidity of tHisH,
which is supported by its resistance to digestion by trypsin (Fig.
3).
The quaternary structure of the tHisH-tHisF complex has to provide the
basis for (a) the activation of the glutaminase reaction at
the active site of tHisH by the binding of PRFAR (or ImGP or ProFAR) to
the active site of tHisF and (b) the transfer of nascent ammonia between tHisH and tHisF in a way that excludes contact with
water, which inevitably would protonate ammonia to the nonreactive ammonium ion. The active site of tHisF is located at the C-terminal face of the central
-barrel (Fig. 4b), but several lines
of evidence suggest that tHisH docks to the N-terminal face of the
barrel. About half of the highly conserved and invariant residues are located close to the N-terminal face of the barrel of tHisF, indicating an important functional role of this region (Fig. 4). Two of these conserved residues, Arg5 and Glu46, were shown
to be important for the glutamine-dependent but not for the
ammonia-dependent reaction of the eHisH-eHisF complex (40).
Furthermore, the region between strand
1 and helix
1, which is located at the C-terminal face of tHisF
(Fig. 4), is not protected by tHisH against trypsinolysis (Fig. 3).
Moreover, docking of tHisH to the C-terminal face of tHisF would impede access of the large substrate PRFAR and should therefore impair catalytic activity. However, both the
KmPRFAR values and the catalytic
efficiencies
kcat/KmPRFAR are
practically identical for isolated tHisF and tHisF in complex with
tHisH (Table I).
How is the binding of PRFAR to tHisF at the C-terminal face of the
-barrel coupled with the glutaminase activity of tHisH, which is
presumably bound to the N-terminal face of the barrel? Arg27 is located in a flexible region between strand
1 and helix
1 at the C-terminal face of
tHisF (Fig. 4). The cleavage of trypsin at Arg27 is
accelerated in the tHisH-tHisF complex compared with isolated tHisF
(Fig. 3). This result indicates that the binding of tHisH to the
N-terminal face of tHisF induces a long range conformational transition
that makes Arg27 more available to proteolytic cleavage.
The amino acid exchange K19S, moreover, which lies in the same flexible
region as Arg27 (Fig. 4), causes larger effects of
kcat and KmPRFAR
in the glutamine-dependent reaction of the tHisH-tHisF
complex (Table IV) than in the ammonia-dependent reaction
of isolated tHisF (Table III). Thus, the flexible region between strand
1 and helix
1 is involved in interactions
between tHisF and tHisH that take place upon the reaction of
nascent ammonia with PRFAR. Since uncharged ammonia is the nucleophile
to attack PRFAR (Fig. 2), ammonia has to be transferred from tHisH to
tHisF without contact with solvent. As a consequence, the transfer will
probably occur through a hydrophobic channel connecting the two active sites, but a high resolution x-ray structure of the tHisH-tHisF complex
is necessary to verify this hypothesis. Ammonia channels of this kind
were recently identified in the GATases carbamoyl phosphate synthase
(4, 41), glutamine phosphoribosyl pyrophosphate amidotransferase (42),
and asparagine synthase B (43).
Plausible Mechanism of the HisF Reaction--
The mechanism of the
cycloligase/lyase HisF reaction (Fig. 2a) is complicated and
unique in primary metabolism (13). Two aspartate residues,
Asp11 and Asp130, are catalytically essential,
since their replacement by asparagines causes an almost complete loss
of tHisF activity (Table III). Saturation random mutagenesis of the
corresponding codons and complementation studies showed that the
function of Asp11 could not be substituted by any other
residue. In contrast, the variant tHisF_D130E complemented a
hisF strain in vivo, albeit more slowly than
wild-type tHisF (Table V). Thus, the restriction for aspartate at
position 130 is not as exclusive as that at position 11, but a
carboxylate side chain appears to be essential at both positions. These
results support the hypothesis that the tHisF reaction depends on
general acid/base catalysis, and a chemically plausible reaction
mechanism was developed on the basis of this finding (Fig.
5). The proposed sequence of reactions
starts with the substitution of the phosphoribulosyl carbonyl oxygen of
PRFAR by ammonia, resulting in the release of water and the
formation of Imine I. In the next step, the addition of
water results in the generation of the first reaction product
AICAR and of Imine II. In the subsequent step,
the imidazole ring of ImGP is closed, and water is released
in a reaction that is catalyzed by the general acid H-A1
and the general base A
. In support of
this mechanism, ProFAR (and probably also PRFAR) are slowly hydrolyzed
in the absence of enzyme to yield AICAR and other unidentified
products, and the rate of this reaction increases with increasing
proton concentration (44). In order to act as a general acid at
physiological pH, the pKa value of an aspartate
within an enzyme must be shifted by several units compared with free
aspartate. Such a shift should be very sensitive to the immediate
environment of the carboxylate function and therefore to the length of
the amino acid side chain. As shown by complementation in
vivo, Asp11 is absolutely required for catalysis and
cannot be functionally replaced by any other amino acid (Table V); it
might therefore be identical with the general acid H-A1.
Asp130 can be functionally replaced by Glu (Table V) and
therefore probably has a more "normal" pKa
value; it might therefore be identical with the general base
A
. The reactions from
PRFAR to Imine I and of Imine I to
Imine II might also be accelerated by general acid/base
catalysis, and Asp11 and Asp130 could play an
essential role here as well. Asp176 could also be involved
in either of the reaction steps, since the kcat
values of the tHisF_D176N variant are decreased about 50-fold compared
with wild-type tHisF (Tables III and IV).

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Fig. 5.
Plausible mechanism of the tHisF reaction
based on general acid/base catalysis. R1,
ribosephosphate, R2, 3-phosphoglycerol. The
general acid H-A1 and the general base
A are probably identical with
Asp11 and Asp130, respectively (see
"Discussion" for details.)
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