Effectors of the Stringent Response Target the Active Site of
Escherichia coli Adenylosuccinate Synthetase*
Zhenglin
Hou
,
Michael
Cashel§,
Herbert J.
Fromm
, and
Richard B.
Honzatko
¶
From the
Department of Biochemistry and
Biophysics, Iowa State University, Ames, Iowa 50011 and the
§ Laboratory of Molecular Genetics, NICHHD, National
Institutes of Health, Bethesda, Maryland 20892
 |
ABSTRACT |
Guanosine 5'-diphosphate 3'-diphosphate (ppGpp),
a pleiotropic effector of the stringent response, potently inhibits
adenylosuccinate synthetase from Escherichia coli as an
allosteric effector and/or as a competitive inhibitor with respect to
GTP. Crystals of the synthetase grown in the presence of IMP,
hadacidin, NO3
, and
Mg2+, then soaked with ppGpp, reveal electron density at
the GTP pocket which is consistent with guanosine 5'-diphosphate
2':3'-cyclic monophosphate. Unlike ligand complexes of the synthetase
involving IMP and GDP, the coordination of Mg2+ in this
complex is octahedral with the side chain of Asp13 in the
inner sphere of the cation. The cyclic phosphoryl group interacts
directly with the side chain of Lys49 and indirectly
through bridging water molecules with the side chains of
Asn295 and Arg305. The synthetase either
directly facilitates the formation of the cyclic nucleotide or
scavenges trace amounts of the cyclic nucleotide from solution.
Regardless of its mode of generation, the cyclic nucleotide binds far
more tightly to the active site than does ppGpp. Conceivably,
synthetase activity in vivo during the stringent response
may be sensitive to the relative concentrations of several effectors,
which together exercise precise control over the de novo
synthesis of AMP.
 |
INTRODUCTION |
Escherichia coli and other bacteria that undergo
nutritional stress, for instance amino acid starvation, synthesize
guanosine 5'-diphosphate 3'-diphosphate
(ppGpp)1 and guanosine
5'-triphosphate 3'-diphosphate (pppGpp). These nucleotides in turn
dramatically influence a broad range of metabolic activities (stringent
response), impairing the synthesis of DNA, rRNA, tRNA, proteins,
nucleotides, and phospholipids and concomitantly stimulating amino acid
biosynthesis and several other functions (1). Evidently the stringent
response stems from a severe shortfall, relative to the demands of
ribosomal protein biosynthesis, in one or more of the aminoacylated
tRNAs. During the stringent response in E. coli, pools of
ATP and GTP decrease, but concentrations of pppGpp and ppGpp rise to
millimolar levels. The reduction in the de novo synthesis of
purine nucleotides putatively is a consequence of direct inhibition of
adenylosuccinate synthetase and IMP dehydrogenase by ppGpp (2-4).
Adenylosuccinate synthetase (IMP:L-aspartate ligase
(GDP-forming), EC 6.3.4.4) is an essential enzyme in E. coli
(2), catalyzing the first committed step in de novo
biosyntheses of AMP as shown in Reaction 1.
The catalytic mechanism is a two-step process (3-5) as follows:
the formation of 6-phosphoryl-IMP by nucleophilic attack of the 6-oxo
group of IMP on the
-phosphoryl group of GTP, followed by a second
nucleophilic displacement of the 6-phosphoryl group by aspartate to
form adenylosuccinate. ppGpp potently inhibits the synthetase
(Km ~50 µM; 6-8). The mechanism of
inhibition by ppGpp, however, remains unsettled. ppGpp may inhibit the
synthetase by binding to an allosteric site, as suggested by its
noncompetitive inhibition with respect to GTP (6, 7), or by simply
binding to the GTP pocket, as suggested by its competitive inhibition with respect to GTP in a different study (8).
Reported here are crystal structures of adenylosuccinate synthetase
from E. coli complexed with IMP,
NO3
, Mg2+, hadacidin, and
guanosine 5'-diphosphate 2':3'-cyclic monophosphate (hereafter the
ppG2':3'p complex), and with IMP, NO3
,
Mg2+, hadacidin, and GDP (hereafter the GDP complex).
Hadacidin, a fermentation product of Penicillium frequentans
(9), is a competitive inhibitor (Ki
~10
6 M) with respect to aspartate (10, 11).
Even though ppGpp was used as a ligand in crystal soaking buffers, the
electron density in the guanine nucleotide pocket is consistent only
with ppG2':3'p. Evidently, ppG2':3'p binds with much greater affinity to the synthetase than does ppGpp. Crystals either absorb a minor impurity of the cyclic nucleotide from solution or the synthetase itself transforms ppGpp to ppG2':3'p. Regardless of its mechanism of
formation, the ppG2':3'p complex here represents the first structure of
an effector of the stringent response with one of its target enzymes.
Results here and from past investigations raise the possibility of
several effectors of the stringent response acting in a coordinated way
to modulate synthetase activity.
 |
MATERIALS AND METHODS |
Purification of Enzyme from E. coli--
The synthetase was
prepared as described previously from a genetically engineered strain
of E. coli (12). The enzyme was at least 95% pure on the
basis of SDS-polyacrylamide gel electrophoresis.
Crystallization--
Hadacidin was a generous gift of Drs. Fred
Rudolph and Bruce Cooper, Department of Biochemistry and Cell Biology,
Rice University. All other reagents came from Sigma. Crystals were
grown by the method of hanging drops under conditions similar to those
employed in previous work (13). For reasons provided below, acetate was excluded from the crystallization droplets, and in preparing crystals for ppGpp soaks, GDP was omitted as well. Droplets (total volume, 12 µl) contained equal parts of an enzyme solution (HEPES (30 mM), IMP (4 mM), hadacidin (4 mM),
GDP (either 0 or 4 mM), and protein (18 mg/ml) at pH 6.8)
and a crystallization buffer (polyethylene glycol 8000 (16% w/v),
cacodylic acid/sodium cacodylate (100 mM, pH 6.5),
magnesium nitrate (100 mM)). The pH of the droplets was 6.5. Wells contained 500 µl of crystallization buffer. Crystals of
approximately 0.8 mm in all dimensions, belonging to the space group
P3221, grew in about 1 week. The omission of GDP and/or acetate had no significant effect on unit cell parameters; the crystalline complexes here are isomorphous to previously published ligand complexes of the synthetase.
Preparation of ppG2':3'p Complex--
ppGpp (preparation 33A)
was prepared as described previously (14). The sample was checked for
purity by thin layer chromatography, using polyethyleneimine plates and
1.5 M KH2PO4, pH 3.4 (14). Synthetase crystals, grown in the absence of guanine nucleotides, were
exposed for 12 h to a solution containing polyethylene glycol 8000 (18% w/v), cacodylic acid/sodium cacodylate (100 mM, pH
6.5), magnesium nitrate (100 mM), IMP (2 mM),
hadacidin (2 mM), and ppGpp (1 mM). A soaked
crystal then was transferred sequentially at 10-min intervals to fresh
solutions, which contained the components above along with glycerol at
concentrations of 5, 10, 15, 20, and 25% (v/v). Glycerol is a
cryoprotectant of adenylosuccinate synthetase crystals. After passing
through the glycerol equilibration steps, a single crystal was mounted
and flash-frozen to 100 K.
Thin Layer Chromatography--
Possible degradation of ppGpp
under conditions employed in soaking crystals was monitored by thin
layer chromatography (14). Aliquots were taken at intervals of 2, 5, 12, and 36 h from a solution containing
Mg(NO3)2 (100 mM), hadacidin (2 mM), cacodylic acid/sodium cacodylate (100 mM,
pH 6.5), and ppGpp (2 mM), spotted on polyethyleneimine
plates, and developed in 1.5 M
KH2PO4 (pH 3.4). Parallel time courses were run
in the absence and presence of adenylosuccinate synthetase at a
concentration of 2 mg/ml (approximately a 40 µM subunit
concentration). GTP and ppGpp were used as standards in thin layer
chromatography. The developed plates were inspected for UV-absorbing
components. A series of solutions, obtained by the dilution of guanine
nucleotide standards of known concentration, were processed by the
chromatographic procedure above, in order to determine the detection
limit for guanine nucleotides.
Data Collection--
Data from single crystals of the GDP
complex and the ppG2':3'p complex were collected on a Siemens area
detector at 100 K and were reduced by XENGEN (15). The data sets were
at least 97% complete to nominal resolutions (at which the average of
I/
(I) is 2) of 2.3 and 2.5 Å for the GDP and ppG2':3'p complexes,
respectively (Table I).
Model Refinement--
Starting phases were calculated from the
GDP complex (13), omitting all ligands and solvent. Refinement
procedures are as described previously (13). The ligand models were fit
to omit electron density maps, followed by a cycle of refinement using XPLOR (16). Constants of force and geometry for the protein came from
Engh and Huber (17), and those for hadacidin from Poland et
al. (13). Refinement parameters for ppG2':3'p were based on those
of GTP and the crystal structures of 2':3'-monophosphate nucleotides
(18-20). In early rounds of refinement, models were heated to 2000 K
and then cooled in steps of 25-300 K. In later rounds of the
refinement, the systems were heated to 1000 or 1500 K and cooled in
steps of 10 K. After the slow-cool protocol was complete (at 300 K),
the models were subjected to 120 steps of conjugate gradient
minimization, followed by 20 steps of individual B-parameter
refinement. Individual B-parameters were subject to the following
restraints: nearest neighbor, main chain atoms, 1.5 Å2;
next-to-nearest neighbor, side chain atoms, 2.0 Å2;
nearest neighbor, side chain atoms, 2.0 Å2; and
next-to-nearest neighbor, side chain atoms, 2.5 Å2.
Criteria for the addition of water molecules were identical to those of
previous studies (5, 13).
 |
RESULTS |
Quality of the Refined Models--
The method of Luzzati (21)
indicates an uncertainty in coordinates of 0.30 Å. The amino acid
sequence used in refinement is identical to that reported previously
(22, 23). Results of data collection and refinement are in Table
I. A overview of the ppG2':3'p complex is
in Fig. 1, and a schematic of ppG2':3'p is in Fig. 2. Gln10 is the
only serious violation of the Ramachandran plot as identified by
PROCHECK (24). The conformation of Gln10 is enforced by its
hydrogen bonding environment as described in previous work (22, 23).
The models have better stereochemistry than is typical for structures
based upon data of comparable resolution. Thermal parameters vary in
the GDP and ppG2':3'p complexes from 10 to 64 Å2 and 10 to
65 Å2, respectively. The variation in thermal parameters
as a function of residue is comparable to that of other structures of
the ligated synthetase (5, 13). Thermal parameters for individual
ligands average to less than 30 Å2 in each of the
structures. Supperposition of the GDP complex onto the ppG2':3'p
complex results in a root mean square deviation of C
coordinates of
0.3 Å, comparable to the estimate of coordinate uncertainty.

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Fig. 1.
Overview of the ppG2':3'p complex of the
synthetase. Stereoview, drawn with MOLSCRIPT (33), of a single
monomer of the synthetase dimer showing IMP, Mg2+, and
ppG2':3'p as ball-and-stick models.
|
|
GDP Complex (Protein Data Base Accession Code
1cib)--
Conditions of crystallization for the GDP complex reported
here differ from those of an earlier study (13) in the elimination of
acetate from the crystallization buffers. Acetate and GDP
synergistically inhibit the synthetase, as does nitrate and GDP (25).
Although the combination of GDP/nitrate exhibits greater synergism than GDP/acetate, the total concentration of acetate in the previous study
was some 40-fold higher relative to the nitrate anion, hence the
potential exists for substantial competition between nitrate and
acetate for the putative
-phosphoryl pocket of GTP. The GDP complex
presented here provides data to higher resolution relative to that of
the previous GDP complex (2.3 as opposed to 2.8 Å), and more
significantly, the active site in the vicinity of the nitrate anion has
stronger electron density and lower thermal parameters. The average
distance of the six oxygen atoms nearest to the Mg2+ (Table
II) in the present GDP complex has
decreased by 0.16 Å relative to the former complex. Most significantly
the distance between Mg2+ and OD1 of Asp13 has
fallen from over 3.0 Å in the GDP complex from acetate buffer to 2.7 Å in the GDP complex without acetate. In the acetate-free buffer, the
synthetase-bound Mg2+ exhibits quasi-octahedral
coordination, rather than the square pyramidal coordination of the
ligated complex in acetate buffer. In addition His41 now
interacts through a bridging water molecule with Glu221,
whereas in the acetate buffer no water molecule is evident, and
His41 exhibits an elevated set of thermal parameters. The
elimination of acetate, then, results in the tighter coordination of
Mg2+ and a more ordered active site in the immediate
vicinity of the
-phosphoryl pocket. As the ppG2':3'p complex
described below comes from an acetate-free buffer, the GDP complex
without acetate reported here serves as a basis of comparison.
ppG2':3'p Complex (Protein Data Base Accession Code 1ch8)--
The
conformation of the synthetase in the ppG2':3'p complex is essentially
identical to that of the GDP complex (Fig.
3 and Table II). Interactions of IMP,
nitrate, and hadacidin are unchanged from previous structures.
Significant differences occur, however, in the ligation of
Mg2+ and at the guanine nucleotide pocket. Within the
uncertainty of the coordinates, Mg2+ exhibits octahedral
coordination, in contrast to the GDP complex, where it exhibits
quasi-octahedral coordination. The oxygen atoms that define the
equatorial plane of the Mg2+ (one each from 5'-
- and
5'-
-phosphoryl groups of ppG2':3'p, from
NO3
, and from the N-formyl
group of hadacidin) average to 2.1 Å, as opposed to 2.3 Å for the
corresponding bonds in the GDP complex (Table II). In the GDP complex,
OD1 of Asp13 tightly hydrogen bonds (2.6 Å) to N-1 of IMP
and is 2.7 Å from the Mg2+. In the ppG2':3'p complex,
however, the distance between OD1 of Asp13 and N-1 of IMP
is 3.1 Å, whereas the distance separating the Mg2+ and OD1
of Asp13 is 2.5 Å. Evidently, the synthetase coordinates
Mg2+ more tightly in the ppG2':3'p complex than in the GDP
complex; the tighter coordination occurring, however, at the expense of a weakened interaction between Asp13 and N-1 of IMP. The
significance of the above to the mechanism of catalysis and inhibition
of the synthetase is discussed below.

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Fig. 3.
Stereoview of interactions between ppG2':3'p
and the active site. Ligands (only hadacidin (Had),
nitrate, ppG2':3'p, and Mg2+ are shown) are drawn with
bold lines. Donor-acceptor interactions (corresponding
distances listed in Table II) are presented with dashed
lines.
|
|
The difference electron density in the guanine nucleotide pocket
clearly indicates an analog of GDP (Fig.
4). Although electron density similar to
that of the GDP complex is present for the base and 5'-pyrophosphoryl
moieties, additional electron density extends from both the 2'- and
3'-hydroxyl groups of the stringent effector. The density can
accommodate a single phosphoryl group but not the pyrophosphoryl group
expected at the 3' position. A difference map, based on observed data
from the effector and GDP complexes, and calculated phase angles from
the GDP complex, reveals a strong and well defined peak of electron
density in the vicinity of the 2'- and 3'-hydroxyl groups of GDP (Fig.
4). The positive difference density overwhelms any negative difference density due to water molecules bound to the 2'- and 3'-hydroxyls of
GDP. Hence the bound nucleotide cannot be GDP. Furthermore, a 3' (or
2')-phosphoryl group cannot occupy the density without causing severe
steric conflict with the remaining 2' (or 3')-hydroxyl group of the
ribose. The only acceptable fit to the electron density was provided by
ppG2':3'p. Thermal parameters for the refined cyclic nucleotide are
comparable to those of the surrounding protein, suggesting full
occupancy of the ligand at the guanosine pocket.

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Fig. 4.
Stereoview of electron density associated
with ligands in the ppG2':3'p complex. A, the electron
density, contoured at 6 using a cut-off radius of 0.9 Å, comes from
a ligand-excised omit map. ppG2':3'p and other active site ligands are
represented with bold lines. B, difference
electron density based on phases from the GDP complex and Fourier
coefficients from the ppG2':3'p and GDP complexes. The contour level is
6 and reveals only residual electron density in the ppG2':3'p
complex that has no counterpart in the GDP complex. The GDP molecule is
represented with bold lines and the ppG2':3'p molecule with
fine lines.
|
|
The synthetase recognizes the 5'-pyrophosphoryl group and the guanine
base of GDP and ppG2':3'p in the same manner. Atom O-6 of the cyclic
nucleotide interacts with OG of Ser414 and backbone amide
331. Endocyclic N-1 and exocyclic N-2 of the base hydrogen bond with
the side chain of Asp333. The 5'-
-phosphoryl group of
ppG2':3'p interacts with backbone amides 15, 16, and 17, the side chain
of His41, and bound Mg2+. The 5'-
-phosphoryl
group interacts with the Mg2+, backbone amide 42, and
Arg305. The ribose moiety of ppG2':3'p, however, differs
significantly in location and conformation relative to that of GDP
(Fig. 4). The 5'-
-phosphoryl group of ppG2':3'p rotates
~120o around the torsion angle defined by atoms PB, OA,
PA, and O-5', displacing the entire ribose moiety approximately 1 Å toward loop 42-53. (Loop 42-53 undergoes a 9-Å conformational change
upon ligation of the synthetase active site.) As a consequence, the cyclic 2':3'-phosphoryl group hydrogen bonds with the side chain of
Lys49 (that side chain is disordered in the GDP complex),
and forms water-mediated hydrogen bonds with the side chains of
Asn295 and Arg305. A water
(Wat605), common to all the ligated structures of the
synthetase, hydrogen bonds to backbone carbonyl groups 42 and 417, and
to atom O-2' of GDP in the GDP complex. In the ppG2':3'p complex O-2'
is now 3.5 Å from the water molecule, bridging backbone carbonyls 42 and 417. The torsion angle O-5'-C-5'-C-4'-C-3' (
by convention) is
77o (-synclinal), the torsion angle
O-4'-C-1
N-9
C-4 (
by convention) is
118o
(anti), both comparable to those of the GDP complex
(
=
66o and
=
82o). The
five-member ring (O-2'-P-1-O-3'-C-3'-C-2') is nearly planar, and
its angles, bond lengths, and ribose ring pucker (C-2'-endo and
C-3'-exo) are comparable to those of cyclic nucleotides, observed in
high resolution crystal structures (19, 20).
Thin Layer Chromatography--
ppG2':3'p is putatively an
intermediate in the degradation of ppGpp (26). ppGpp is acid- and
alkali-labile but relatively stable at neutral pH. The stability of
ppGpp was monitored by thin layer chromatography in solutions of
composition indicated under "Materials and Methods." No detectable
hydrolysis product of ppGpp appeared up to 36 h in aliquots
without adenylosuccinate synthetase. Nor did a hydrolysis product
appear under the same conditions in the presence of the synthetase at a
concentration of 2 mg/ml. The minimum concentration of guanine
nucleotide detectable by the chromatographic protocol is approximately
80 µM. Hence hydrolysis products of ppGpp could be
present in the original sample or any of the solutions used to monitor
the hydrolysis of ppGpp at a level of up to 4 parts per 100 and still
escape detection.
 |
DISCUSSION |
Under the conditions of our crystallographic studies ppGpp does
not undergo significant hydrolysis. Two possibilities, then, can
account for the appearance of ppG2':3'p in the guanine pocket of the
synthetase: either the crystalline synthetase scavenges a minor (and
undetectable) impurity of the cyclic nucleotide from our sample of
ppGpp or the synthetase itself facilitates the conversion of ppGpp to
the cyclic nucleotide. As no build-up of the cyclic nucleotide was
observed in the presence of the synthetase, the putative
enzyme-mediated process must occur without appreciable turnover. For
the latter scenario, ppGpp must bind to the guanine pocket and undergo
cyclization, producing an inhibitor that does not readily dissociate
from the synthetase.
As a means of better understanding a possible enzyme-mediated
mechanism, one can model a transition state complex for the cyclization
of ppGpp in the guanine nucleotide pocket (Fig.
5). Mg2+ (total concentration
of 100 mM) must be associated with the 3'-pyrophosphoryl group of ppGpp under the conditions of crystal soaks. The water molecule, which bridges backbone carbonyls 42 and 417 and hydrogen bonds with the 2'-hydroxyl of GDP (Table II), may act as a weak catalytic base and abstract a proton from the 2'-OH of ribose (Fig. 5).
The activated O-2' atom then attacks the 3'-
-phosphoryl group,
displacing the 3'-
-phosphoryl group. The side chain of Lys49, as well as the Mg2+ associated with the
3'-pyrophosphoryl group, would stabilize the development of negative
charge on the leaving group and/or the transition state. Lysyl side
chains in analogous positions are critical to the catalytic function of
adenylate kinase and GTPases in general.

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Fig. 5.
Stereoview of a model for the putative
transition state in the conversion of ppGpp into ppG2':3'p at the
active site of the synthetase. Ligands (hadacidin
(Had), Mg2+, and the nucleotide transition
state) are drawn with bold lines. Bold, dashed
lines represent the geometric relationship of the attacking
nucleophile and leaving group with respect to the 3'- -phosphoryl
group. Possible donor-acceptor interactions are represented with
dashed lines. A second Mg2+, which could
interact with the 3'-pyrophosphoryl group of the nucleotide, is not
shown.
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|
Regardless of its mode of formation, ppG2':3'p must bind at least
200-fold more tightly than does ppGpp to the active site of the
synthetase, assuming the cyclic nucleotide is 4% of the concentration
of ppGpp in soaking solutions and that the Ki for
ppGpp is 50 µM. The estimated Ki for
ppG2':3'p (~10
7 M) compares favorably to
the tightest known inhibitors of the synthetase, alanosyl-AICOR
(~10
7 M (27)) and hydantocidin 5'-phosphate
(~10
8 M (28)). The enhanced binding of
ppG2':3'p may arise from the following three sources. (i) Cyclization
of the 2'- and 3'-hydroxyls of the ribose reduces conformational
freedom of the nucleotide and hence reduces the entropic penalty
associated with ligand binding. (ii) The 2':3'-phosphoryl group
provides additional opportunities for strong hydrogen bonds with the
synthetase. (iii) The active site Mg2+ has octahedral
coordination, whereas in GDP complexes its coordination is square
pyramidal (13) or quasi-octahedral (see above). In fact, an
octahedrally coordinated metal cation appears as well in the complex of
the tight binding inhibitor hydantocidin 5'-phosphate (29).
Asp13 interacts directly with IMP in GDP complexes (see
above) and evidently in the absence of an IMP-Asp13
interaction (as in the hydantocidin 5'-phosphate complex (29)) or a
weakened IMP-Asp13 interaction (as here in the ppG2':3'p
complex), Asp13 favors direct coordination to the
Mg2+. Octahedral coordination of the active site
Mg2+, then, may be a hallmark of tight binding inhibitors
of the synthetase. Such inhibitors are of potential significance in
chemotherapy and the development of herbicides (27, 28).
A cyclic nucleotide at the guanine nucleotide pocket does not explain
competitive inhibition between ppGpp and IMP and noncompetitive inhibition between ppGpp and GTP, as observed in two investigations (6,
7). Such phenomena could arise from impure preparations of the
synthetase (8). Alternatively, ppGpp could bind to a site distinct from
the guanine nucleotide pocket. Synthetase dimers, for instance,
putatively bind IMP with an affinity 100-fold greater than that of
synthetase monomers. Hence, ppGpp will inhibit competitively with
respect to IMP and non-competitively with respect to GTP, by binding to
a site that blocks dimerization of the synthetase. The existence of
such a site is inferred by covalent modification of the synthetase by
pyridoxal phosphate (30) and
guanosine-5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate) (31) at a cluster of basic residues, exposed in synthetase monomers, but buried between monomers in the synthetase dimer. The experimental protocol developed here reveals ppGpp interactions at the guanine nucleotide pocket (consistent with all kinetics investigations (6-8))
but does not probe for a ppGpp-binding site at the buried interface
between subunits of the synthetase dimer.
Potent inhibition of adenylosuccinate synthetase in vivo
typically impedes growth of the organism. The pur
A
, E. coli cell line grows poorly, even
in rich medium. Hydantocidin (which is transformed in plants to the
tight binding synthetase inhibitor, hydantocidin 5'-phosphate) is a
potent herbicide (27). Alanosyl-AICOR and hadacidin are antibiotics (9,
28), the latter having only the synthetase as a known target. Hence, if the stringent response is ultimately a coordinated attempt by E. coli to limit its own growth under unfavorable conditions, then
the synthetase is an appropriate target.
Even though ppGpp is an effective inhibitor of the synthetase at its
physiological concentration during the stringent response, other
effectors (pppGpp and ppG2':3'p) may also influence synthetase activity. The transformation of ppGpp into ppG2':3'p, perhaps at the
active site of the synthetase, suggests a parallel and more potent
mechanism of inhibition, which may be temporally distinct from the
inhibition by ppGpp. pppGpp, putatively the first nucleotide generated
during the stringent response, could be an inhibitor of the synthetase,
its 5'-
-phosphoryl group being transferred to IMP while the
cyclization reaction occurs at the guanine pocket. The result would be
an active site complex of 6-phosphoryl-IMP and ppG2':3'p. The
combination of the synthetase with GTP and 6-thio-IMP has resulted in
the formation of a stable complex of 6-thiophosphoryl-IMP at the active
site (5). Furthermore, the combination of IMP and GTP leads to the
analogous 6-phosphoryl-IMP complex (32). Hence, the double chemical
transformation of pppGpp at the active site of the synthetase is well
within the realm of possibility. Alternatively, if pppGpp is a simple
substrate of the synthetase, then in vivo inhibition would
depend on the relative concentrations and affinity constants of pppGpp
and ppGpp, as well as the concentration of the cyclic effector. Hence
the inhibition of the E. coli synthetase by stringent
effectors may be far more complex than the competition of ppGpp for the
active site.
 |
FOOTNOTES |
*
This work was supported in part by Grant NS 10546 from the
National Institutes of Health, the United States Public Health Service,
Grant MCB-9603595 from the National Science Foundation, and Grant
95-37500-1926 from the United States Department of Agriculture. This is
Journal Paper J18338 from the Iowa Agriculture and Home Economics
Experiment Station, Ames, IA, Project 3191, and is supported by Hatch
Act and State of Iowa funds.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.
The atomic coordinates and structure factors (codes 1cib and
1ch8) have been deposited in the Protein Data Bank, Brookhaven National
Laboratory, Upton, NY.
¶
To whom correspondence should be addressed: Dept. of
Biochemistry and Biophysics, Iowa State University, Ames, IA 50011. Tel.: 515-294-6116; Fax: 515-294-0453; E-mail:
honzatko{at}iastate.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
ppGpp, guanosine
3',5'-bis(diphosphate), pppGpp, guanosine 3'-diphosphate
5'-triphosphate, ppG2':3'p, guanosine 5'-diphosphate 2':3'-cyclic monophosphate;
alanosyl-AICOR, l-alanosyl-5-amino-4-imidazolecarboxylic
acid ribonucleotide.
 |
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