From the Unité Mixte de Recherche 1932, Laboratoire Mixte CNRS/Institut National de la Recherche Agronomique/Bayer Cropscience, 14-20 Rue Pierre Baizet, 69263 Lyon, France
Received for publication, July 23, 2002, and in revised form, October 14, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The regulatory domain of the bifunctional
threonine-sensitive aspartate kinase homoserine dehydrogenase contains
two homologous subdomains defined by a common loop- In plants and bacteria, the first and the third steps of
methionine and threonine biosynthesis are catalyzed by isoforms of the
bifunctional enzyme, aspartate kinase (AK)1
(EC 2.7.2.4)-homoserine dehydrogenase
(HSDH) (EC 1.1.1.3) (1, 2). In Escherichia coli, the
expression of one of these bifunctional enzymes is repressed by
methionine (3), whereas the expression of the other enzyme is repressed
by threonine (4). The activity of this later bifunctional isoform is
also inhibited by threonine. The threonine-sensitive isoform has been
extensively characterized in bacteria (5, 6). It is a homotetramer
containing two threonine-binding sites per monomer (5, 6). Although the
two threonine-binding sites of the bacterial enzyme have not yet been
identified, preliminary work has demonstrated that the regulatory
domain of the bacterial enzyme is located in the intermediary region
(amino acids segment 316-447) between the AK catalytic domain (amino
acids segment 1-315) and the HSDH catalytic domain (amino acids
segment 448-812) (7-11) (Fig. 1). Furthermore, internal sequence
comparisons carried out on the E. coli enzyme indicated that
the intermediary region corresponding to the regulatory domain is
composed of two homologous subdomains (316-366 and 397-447) sharing
47% of amino acid identities (9) (Fig. 1).
In plants, two genes encoding bifunctional AK-HSDH were also found
(12-15). The corresponding isoforms exhibit high homologies with the
bacterial threonine-sensitive AK-HSDH. In Arabidopsis thaliana, the genes encoding the two AK-HSDH are located on
chromosome 1 (13) (GenBankTM accession number
trEMBL Q9SA18) and on chromosome 4 (GenBankTM
accession number trEMBL O81852). As a first step toward a
structural characterization of the bifunctional threonine-sensitive AK-HSDH in plants, we reported previously (16) on the purification to
homogeneity of one isoform from A. thaliana (trEMBL O81852). In the present paper the organization of the regulatory domain of
A. thaliana AK-HSDH is examined. Comparisons of predicted
secondary structures of the biosynthetic threonine deaminase (TD) and
AK-HSDH as well as the knowledge of TD three-dimensional structure (17, 18) allowed us to point to two amino acids in the AK-HSDH regulatory domain sequence potentially involved in the two threonine-binding sites
of the enzyme. These amino acids were mutated, and the kinetic behavior
of the mutants was determined and compared with that of the wild-type
enzyme. Results indicate that the position of the amino acid in the
threonine-binding sites was predicted correctly. Moreover, steady-state
kinetics allow us to propose a structural model for the mechanism of
control of AK and HSDH activities by threonine.
Materials--
New England Biolabs supplied restriction
endonucleases. Isopropyl Site-directed Mutageneses--
Site-directed mutageneses were
carried out on the previously constructed pET23/AK-HSDH vector (16)
using the QuickChangeTM site-directed mutagenesis kit
(Stratagene). Oligonucleotides were designed to replace
Gln443 and Gln524 of the A. thaliana
AK-HSDH by alanine and to modify the restriction enzyme digestion
profile for identification of mutants. Sequencing of the mutants was
performed (Genome Express) and showed no mutations other than those desired.
Overproduction and Purification of AK-HSDH--
Overproduction
and purification of the A. thaliana mutants were carried out
as published previously for the wild-type AK-HSDH enzyme (16).
In Vitro Assays of AK--
AK activity was assayed in the
forward direction by the hydroxamate method (19). Enzyme activity was
expressed as micromoles of aspartyl-P produced per min In Vitro Assays of the Reverse Reaction of HSDH--
Aspartate
semialdehyde is not available as a commercial product. Therefore, HSDH
activity was first assayed in the nonphysiological direction (aspartate
semialdehyde synthesis from homoserine and NADP+). Enzyme
activity was expressed as micromoles of NADPH produced per
min Production of Aspartate Semialdehyde--
HSDH activity was also
measured in the forward direction. In this case, aspartate semialdehyde
was produced from aspartate and ATP using monofunctional aspartate
kinase and aspartate semialdehyde dehydrogenase (EC 1.2.1.11).
Aspartate semialdehyde was produced extemporaneously in a medium
containing 50 mM Tris-HCl, pH 8.0, 100 mM
aspartate-KOH, pH 8.0, 50 mM ATP-KOH, pH 8.0, 50 mM MgCl2, 50 mM KCl, 4 mM NADPH, and 100 mM L-malate-KOH,
pH 8.0. Production of aspartate semialdehyde was initiated by the
addition of 30 µg of pure A. thaliana monofunctional AK
(overproduced and purified in our
laboratory)2 and 70 µg of
pure A. thaliana aspartate semialdehyde dehydrogenase (20).
Regeneration of NADPH was driven by the addition of 25 µg of
commercial chicken liver malic enzyme (EC 1.1.1.40). The reaction was
allowed to proceed for 2 h at 37 °C in a volume of 250 µl.
Protein was then eliminated by centrifugation on Nanosep 10K (Pall
Filtron). Aspartate semialdehyde concentration in the eluate was then
determined enzymatically with an excess of NADPH and AK-HSDH.
In Vitro Assays of HSDH in the Forward Direction--
Enzyme
activity was expressed as micromoles of NADPH transformed per
min Gel Filtration Experiments--
Molecular mass determinations
were carried out on a HiLoad 16/60 Superdex S 200 (Amersham
Biosciences) column equilibrated in 50 mM Hepes-KOH, pH
7.5, 150 mM KCl, and 10% glycerol (v/v) with (5 mM) or without threonine.
Electrophoresis and Protein Determination--
SDS-PAGE were
performed according to Chua (21). Protein concentration was measured
either by the method of Bradford (22) (for crude extracts only) with
bovine Kinetic Data Analyses--
Kinetic data were fitted with the
appropriate theoretical equations by using the KaleidaGraph program
(Abelbeck software).
Secondary Structure Analyses--
Secondary structure analyses
were carried out with the program SOPM available on the Expasy web site
(24). A search of homologies was carried out with the Pfam protein data
base (25).
Regulatory Domain of AK-HSDH--
It was previously shown by
internal sequence comparison that the E. coli
threonine-sensitive AK-HSDH regulatory domain is composed of two
homologous subdomains (9). This feature is also found for A. thaliana threonine-sensitive AK-HSDH (Fig.
1). Indeed, subdomain 1 (residues
414-453) and subdomain 2 (residues 495-534) of the plant AK-HSDH
exhibit 33% of identity at the amino acid level (Fig. 1). To
characterize more deeply the regulatory domain of the plant enzyme, and
in particular the sites where threonine potentially binds, we
analyzed the predicted secondary structure of the subdomains. As shown
in Fig. 1, each subdomain of the regulatory domain of AK-HSDH is
predicted to exhibit a common loop- Comparisons with TD--
The primary sequence of the regulatory
domain of TD does not exhibit homology with that of the regulatory
domain of AK-HSDH. However, the structure of the E. coli TD
crystallized in the absence of effector (17) clearly shows that the
regulatory domain of TD is also composed by a pair of
loop- Effector-binding Sites of TD--
In our study of A. thaliana TD we showed that each subdomain of the regulatory domain
of TD possesses an effector-binding site (18). Indeed, the amino acid
residue located at the C-terminal extremity of the first Prediction of the Amino Acid Residues Involved in AK-HSDH
Threonine-binding Sites--
Because of the structural similarity
between TD and AK-HSDH regulatory domains, the locations of the
putative amino acids involved in the threonine-binding sites in AK-HSDH
regulatory domain can thus be predicted. The putative amino acids
selected for mutagenesis studies were chosen as follows. First, the
amino acids selected must be conserved between subdomain 1 and
subdomain 2 of the regulatory domain of AK-HSDH. Second, these amino
acids must be located at the end of the first Construction, Overproduction, and Purification of AK-HSDH
Mutants--
Site-directed mutageneses were carried out on vector
pET23/AK-HSDH (16) and two mutants were constructed
(pET23/AK-HSDH(Q443A) and pET23/AK-HSDH(Q524A)). Overproduction and
purification to homogeneity were carried out for each mutant as
described for the wild-type A. thaliana enzyme (16).
Solubility and yield of the two purified mutants were similar to those
obtained for the wild-type AK-HSDH (16) (not shown). For each mutant, a
K0.5 value for threonine was determined for the
inhibition of AK and HSDH activities. Kinetic parameters for AK
activity and both directions of HSDH reversible reaction were also determined.
Inhibition of AK Activity by Threonine--
In the wild-type
enzyme the inhibition of AK activity by threonine originates from an
increase in the apparent Km values for ATP and
aspartate (16). At saturation of aspartate and ATP, threonine inhibits
AK activity (Fig. 3A) in a
cooperative manner (K0.5 = 500 µM;
nH of 1.95). Inhibition is virtually 100% at
saturation of threonine. Fig. 3A shows that the mutation of the residue in subdomain 1 (Q443A) or in subdomain 2 (Q524A) leads to
different effects on the inhibition pattern. AK activity of the mutant
Q443A becomes completely insensitive to threonine inhibition (Fig.
3A). By contrast, mutation of residue Gln524
does not modify the K0.5 value for inhibition of
the activity of AK by threonine (K0.5 = 368 µM) (Fig. 3A).
Inhibition of HSDH Activity by Threonine in the Reverse
Direction--
We demonstrated previously for the wild-type enzyme
(16) that the inhibition of HSDH activity by threonine in the reverse direction can be fitted by a hyperbolic equation (see Fig.
3B). The concentration of threonine required to inhibit 50%
of wild-type HSDH activity in the reverse direction is high
(K0.5 = 60 mM) compared with that
required for 50% inhibition of wild-type AK activity
(K0.5 = 500 µM). As reported
previously, the inhibition of wild-type HSDH activity in the reverse
direction results from an increase in the apparent
Km values for NADP+ and homoserine (16).
At saturation of threonine a maximum inhibition of 85% of the reverse
reaction of HSDH activity is reached. Fig. 3B shows that
mutation of the residue in subdomain 1 (Q443A) or in subdomain 2 (Q524A) leads to similar effects on the inhibition of HSDH activity in
the reverse direction by threonine. Indeed, the
K0.5 value for inhibition of the HSDH reverse
reaction by threonine was increased by a factor of approximately 6 for
both mutants Q443A (K0.5 = 347 mM)
and Q524A (K0.5 = 335 mM) (Fig. 3B). This contrasts with the results obtained when AK
activity of these mutants was measured (only mutation Q443A led to
modification of the inhibition pattern). Although the
K0.5 value for inhibition by threonine of the
reverse reaction of HSDH does not correspond to a physiological value,
mutations of Gln443 and Gln524 led in both
cases to an alteration of the apparent affinity for threonine. These
results clearly demonstrate the existence of two nonequivalent
threonine-binding sites per monomer.
Inhibition of HSDH Activity by Threonine in the Forward
Direction--
To characterize the inhibition of HSDH activity in the
forward direction, aspartate semialdehyde was produced using purified monofunctional aspartate kinase and aspartate semialdehyde
dehydrogenase (described under "Experimental Procedures"). Fig.
3C shows that the inhibition of the forward reaction of
wild-type HSDH activity by threonine can be fitted by a hyperbolic
equation. A K0.5 value of 12 mM was
calculated. This value is 5-fold lower than that obtained
for the reverse reaction (Fig. 3C). As observed for HSDH activity in the reverse direction, threonine does not lead to a
complete inhibition (80% of inhibition) of the forward reaction of the
wild-type HSDH. Mutation of residues from subdomain 1 (Q443A) or
subdomain 2 (Q524A) leads to a similar decrease in the apparent affinity for threonine. Indeed, the K0.5 value
for inhibition of HSDH activity in the forward direction was increased
by a factor of approximately 10 for both mutants. A value of 119 mM was indeed calculated for the Q443A mutant and 92 mM for the Q524A mutant (Fig. 3C). The same
conclusions as those given above for the reverse direction can thus be
drawn from the more delicate measurements of the reaction in the
forward direction.
Kinetic Parameters for AK and HSDH Activities--
The effects of
the mutations on the kinetic parameters (Vmax,
Km) were also determined for each mutant. The
maximal velocity of AK and HSDH reactions was not modified for mutants Q443A and Q524A compared with that determined for the wild-type enzyme
(see Tables I and
II). The effects of the mutations on the
Km values of substrates and cofactors of both AK and
HSDH were also determined. As shown in Table I, mutation of
Gln443 or Gln524 does not modify the kinetic
parameters of the forward and reverse reactions of HSDH. By contrast
and as shown in Table II, the kinetic properties of AK for aspartate
and ATP were found to be slightly modified for mutants Q443A and Q524A
compared with the wild-type enzyme. Indeed, the Km
value for aspartate was increased by factors of 2 and 5 for mutants
Q443A and Q524A, respectively. The Km value for ATP
was decreased by a factor of approximately 5 for mutants Q443A and
Q524A.
Gel Filtration Experiments--
We observed previously that
elution of the wild-type AK-HSDH on Superdex S 200 (HiLoad
1.6 × 60) is modified on addition of threonine (16). Indeed, when
gel filtrations were carried out without threonine, wild-type AK-HSDH
was eluted with an apparent molecular mass of 470 kDa (16)
corresponding to an oligomer with a size larger than that of a tetramer
(Fig. 4). However, in the presence of 5 mM threonine, the wild-type enzyme behaved as a tetramer
with an apparent molecular mass of 320 kDa (16). To determine whether
threonine had an effect on the quaternary structure of the AK-HSDH
mutants, gel filtration experiments were carried out with the mutant
enzymes in the presence or the absence of the effector. By contrast
with the wild-type enzyme, elution of mutants Q443A and Q524A is not
modified by the addition of threonine (5 mM) (Fig. 4).
Indeed, the mutant enzymes were eluted with an apparent molecular mass
of 470 kDa with or without threonine (Fig. 4).
Identification of Two Nonequivalent Threonine-binding
Sites--
Previous binding experiments have demonstrated that each
monomer of AK-HSDH is able to bind two threonine molecules (5, 6).
However, the location of the two binding sites was unknown. Our work
allowed the identification of two amino acid residues (Gln443 and Gln524) belonging to two
threonine-binding sites on each monomeric unit of threonine-sensitive
AK-HSDH. Kinetic measurements of threonine inhibition on mutants Q443A
and Q524A demonstrated clearly that these two sites are nonequivalent.
Indeed, mutation of Gln443 changes the inhibition by
threonine for both AK and HSDH activities, whereas mutation of
Gln524 only alters the inhibition by threonine for HSDH activities.
Proposed Model for the Mechanism of Inhibition by
Threonine--
Kinetic experiments showed that mutation of
Gln524 in subdomain 2 leads to an increase in the
K0.5 threonine value for inhibition of HSDH
activities (forward and reverse directions). This result suggests
therefore that binding of threonine on subdomain 2 is involved in the
inhibition of HSDH activity (Fig. 5).
Furthermore, because the mutation of subdomain 2 does not modify the
K0.5 threonine value for inhibition of AK
activity, this result also indicates that binding of threonine on
subdomain 2 controls exclusively HSDH activity. Analysis of
kinetic experiments carried out on mutant-modified subdomain 1 is more
complex. Indeed, modification of subdomain 1 (Q443A) completely
suppressed the ability of threonine to inhibit AK (Fig. 3). This result
suggests that binding of threonine on subdomain 1 is responsible for
inhibition of AK activity (Fig. 5). Mutagenesis of subdomain 1 also
induces a modification of the K0.5 threonine for
inhibition of HSDH activities. Because HSDH inhibition was shown to be
dependent on threonine binding on subdomain 2, an interpretation of
this result is that in addition to preventing threonine binding on
subdomain 1, mutation of subdomain 1 also hinders the binding of
threonine on subdomain 2. Thus, a coupling between subdomain 1 and
subdomain 2 exists, but the communication is not reciprocal. From the
observations of the effect of threonine on the kinetics of wild-type
and mutant enzymes, one can propose that Gln443 is a high
affinity binding site for threonine, whereas Gln524 is a
low affinity binding site for the effector. Our results suggest
therefore that interaction of threonine with the high affinity binding
site Gln443 would lead to a loss of AK activity (Fig. 5).
At the same time, threonine interaction with this high-affinity site
would also induce a modification of the conformation of subdomain 2, allowing the second binding site with lower affinity
(Gln524) to interact with a second threonine (Fig. 5). The
binding of this second threonine would lead to the inhibition of HSDH
activity (Fig. 5). Final validation of this model will be obtained with the determination of crystallographic structure of the wild-type and
mutant enzymes with and without threonine.
Comparison with the Mechanism of Control of Threonine Deaminase by
Isoleucine--
In a previous study that combined kinetic and binding
experiments we showed that subdomain 1 of TD (containing
Tyr449) is a high affinity binding site for isoleucine,
whereas subdomain 2 (containing Tyr543) is a low-affinity
binding site for isoleucine (18). It was also demonstrated that
interaction of subdomain 1 of TD with a first isoleucine induces 1) a
modification of conformation of the catalytic domain of TD leading to a
slight activation of enzyme activity, and 2) a conformational
modification of subdomain 2 leading to an enhancement of the affinity
of subdomain 2 for isoleucine. Finally, isoleucine interaction with
subdomain 2 induces conformational modifications of the catalytic
domain leading to final inhibition of the enzyme (18).
Our present results on AK-HDSH mutants suggest therefore that the
mechanism of control of AK-HSDH by threonine is similar to the
mechanism of control of TD by isoleucine as described above. Indeed,
for AK-HSDH and TD, mutation of subdomain 2 leads only to a decrease in
the affinity of subdomain 2 for the effector, whereas mutation of
subdomain 1 leads to a decrease in the affinity of both subdomains for
the effector. Thus, one can propose that in the wild-type AK-HSDH and
TD, interaction of a first effector on subdomain 1 induces
conformational modification leading to or facilitating the binding of a
second effector on subdomain 2 (Fig. 5).
Gel Filtration Analysis--
In parallel to the loss of
sensitivity toward threonine inhibition, the apparent molecular mass of
mutants Q443A and Q524A also becomes insensitive to threonine addition
(at the concentration of threonine used in the experiment). This result
shows that binding of threonine on the wild-type AK-HSDH induces
conformational modifications that cannot occur in the mutant enzymes.
Because the Q524A mutant still binds threonine on subdomain 1 (Gln443), one can propose that binding of threonine in
subdomain 2 is responsible for the shift in the apparent molecular
weight observed when wild-type enzyme is gel-filtered in the presence
of threonine. Further work is required to characterize the effect of
threonine binding on the quaternary structure of the enzyme.
Amino Acids Involved in the Effector-binding Sites--
As
described above, the amino acid residues located between the C-terminal
extremity of the first
As observed for mutant Q443A, mutation of Ile442 (and not
Ile522) by alanine leads to a loss of threonine sensitivity
for the AK activity (results not shown). In agreement with the behavior
of the mutants Q443A and Q524A, mutation of Ile442 and
Ile522 by alanine leads to a decrease of threonine
sensitivity for inhibition of HSDH activities (results not shown).
Additional mutations and crystallographic determination of the
structure in the presence of the effector will be required to define
accurately all the amino acid residues conferring the specificity of
the effector-binding site of each subdomain.
General Implication and Prediction--
A main issue of the
characterization and comparison of the different mechanisms of control
of allosteric enzymes is to determine whether general mechanisms can be
uncovered and therefore used to predict function from the analysis of
the amino acid sequences. To determine whether the
loop-
The search showed that this motif is annotated as an "ACT domain."
Interestingly and as described recently (26), this motif can be found
in a great many allosteric proteins involved in amino acid or purine
biosynthesis. As shown in the Pfam protein families data base (25) and
as reported recently (26), the ACT domain can be found in one copy or
in duplicate (in the case of AK-HSDH or TD). As demonstrated by the
work carried out on TD (18) and AK-HSDH (this article) the existence of
a pair of ACT domains would lead to the creation of two nonequivalent
binding sites allowing complex regulatory patterns of various protein activities.
helix-loop-
strand-loop-
strand motif.
This motif is homologous with that found in the two subdomains of the
biosynthetic threonine-deaminase regulatory domain. Comparisons of the
primary and secondary structures of the two enzymes allowed us to
predict the location and identity of the amino acid residues
potentially involved in two threonine-binding sites of
Arabidopsis thaliana aspartate kinase-homoserine
dehydrogenase. These amino acids were then mutated and activity
measurements were carried out to test this hypothesis. Steady-state
kinetic experiments on the wild-type and mutant enzymes demonstrated
that each regulatory domain of the monomers of aspartate
kinase-homoserine dehydrogenase possesses two nonequivalent
threonine-binding sites constituted in part by Gln443 and
Gln524. Our results also demonstrated that threonine
interaction with Gln443 leads to inhibition of aspartate
kinase activity and facilitates the binding of a second threonine on
Gln524. Interaction of this second threonine with
Gln524 leads to inhibition of homoserine dehydrogenase activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactoside was
supplied by Roche Molecular Biochemicals and amino acids by
Sigma. Oligonucleotides used for PCR amplifications and site-directed
mutageneses were obtained from Genome Express (Meylan, France). Chicken
liver malic enzyme (EC 1.1.1.40) was purchased from Sigma.
1
per mg
1 of protein using an
505 nm of 750 M
1cm
1. The assay mixture
contained 100 mM Tris-HCl, pH 8.0, 400 mM hydroxylamine-KOH, pH 8.0, 0-50 mM aspartate-KOH, pH 8.0, 0-40 mM ATP-KOH, pH 8.0, 20 mM
MgCl2, and 150 mM KCl. The reaction was
initiated by the addition of AK-HSDH enzyme (final volume, 1 ml) and
was carried out at 37 °C for 10 min. The reaction was terminated by
the addition of a 0.5-ml solution of 0.37 M
FeCl3, 20% trichloroacetic acid, and 0.72 M HCl. The mixture was centrifuged for 15 min at
10,000 × g, and the absorbance of the supernatant was
measured at 505 nm.
1 per mg
1 of protein using an
340 nm of 6250 M
1·cm
1. The mixture contained
100 mM Tris-HCl, pH 8.0, 150 mM KCl, 0-1 mM NADP+, and 0-50 mM homoserine.
The reaction was initialized by the addition of AK-HSDH enzyme (final
volume, 1 ml) and was carried out at 37 °C. Enzyme activity was
visualized by monitoring absorbance changes at 340 nm.
1 per mg
1 of protein using an
340 nm of 6250 M
1·cm
1. The mixture contained
100 mM Tris-HCl, pH 8.0, 150 mM KCl, 0-1 mM NADPH, and 0-5 mM aspartate semialdehyde.
The reaction was initiated by the addition of AK-HSDH enzyme (final
volume, 250 µl) and was carried out at 37 °C. Enzyme activity was
visualized by monitoring absorbance changes at 340 nm.
-globulin as standard or by measuring
A205 as described by Scopes (23).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helix-loop-
strand-loop-
strand motif
(loop-
-loop-
-loop-
motif).
View larger version (22K):
[in a new window]
Fig. 1.
Regulatory domain of AK-HSDH. A,
functional domains of AK-HSDH. The drawing corresponds to the sequence
of the plant enzyme. B, comparisons of the primary and
secondary structures of the two subdomains of the regulatory domain of
A. thaliana AK-HSDH. Sd 1, subdomain 1; sd
2, subdomain 2. Conserved amino acids are indicated in
bold. helices (h),
strands
(e), and loops (coils (c) or turns
(t)) are colored. C, motif
corresponding to the secondary structure of each subdomain.
D, structure of the regulatory domain of the TD enzyme from
E. coli (1TDJ, Refs. 17 and 18). The regulatory domain is
made of two homologous subdomains. The regulatory domain of the plant
enzyme has a similar fold. Amino acid residues (Tyr369 and
Tyr465 for E. coli, Tyr449 and
Tyr543 for A. thaliana) involved in the two
nonequivalent binding sites are indicated. Secondary structures found
in the motif of each subdomain are colored. Parts of the
regulatory domain not involved in the loop-
-loop-
-loop-
motif
are represented in gray.
-loop-
-loop-
motifs as predicted in the AK-HSDH
regulatory domain (Fig. 1). Thus, although the primary sequences of TD
and AK-HSDH are different, the regulatory domains of these enzymes
probably have more similar folds (Fig. 2).
View larger version (34K):
[in a new window]
Fig. 2.
Primary and secondary structures of
the subdomains of the regulatory domain of AK-HSDH and TD.
A, comparison of primary and secondary structures between
the two subdomains of the regulatory domain of A. thaliana
TD. Amino acid residues (Tyr449 and Tyr543)
involved in the two effector-binding sites of TD are
underlined. Sd 1, subdomain 1; sd 2,
subdomain 2. B, comparison of primary and secondary
structures between the two subdomains of the regulatory domain of
A. thaliana AK-HSDH. Amino acid residues investigated by
site-directed mutagenesis are underlined. C,
comparison of secondary structures of the two subdomains of the
regulatory domain of AK-HSDH and TD. Conserved amino acids are
indicated in bold. helices (h),
strands
(e), and loops (coil (c) or turn (t))
are indicated.
strand (underlined in the motif) of each loop-
-loop-
-loop-
motif belongs to an
effector-binding site (Figs. 1 and 2) (18). These amino acid residues
are Tyr449 and Tyr543 in A. thaliana TD (Figs. 1 and 2) (18).
strand of the
loop-
-loop-
-loop-
motif found in each subdomain. We selected
at this step three amino acids in each subdomain. These amino acids
were Ile441, Ser442, and Gln443 for
subdomain 1 and Ile522, Ser523, and
Gln524 for subdomain 2. Third, the amino acids selected
must be conserved between plant and bacterial threonine-sensitive
AK-HSDH sequences. From this last selection, only Ile441
and Gln443 for subdomain 1 and Ile522 and
Gln524 for subdomain 2 are conserved between various plants
and bacterial threonine-sensitive AK-HSDH. Therefore, these amino acids
were selected for mutagenesis and replacement by alanine residues. Mutation of Ile or Gln of each subdomain leads to large modifications of the threonine sensitivity of the mutant enzymes. However, the strongest effects were observed for Gln443 and
Gln524 mutants. As a consequence, we choose to report under
"Results" only data concerning the mutagenesis of
Gln443 and Gln524.
View larger version (18K):
[in a new window]
Fig. 3.
Effect of threonine on AK and HSDH activities
of the wild-type and mutant AK-HSDH enzymes. A, effect on AK
activity. The data corresponding to the wild-type enzyme and to mutant
Q524A were fitted with a Hill equation (wild type (WT)
K0.5 = 0.50 ± 0.01 mM,
nH = 1.95 ± 0.08; Q524A,
K0.5 = 0.37 ± 0.01 mM,
nH = 2.14 ± 0.07). Mutant Q443A is
completely insensitive to threonine inhibition (up to 100 mM threonine). B, effect on the reverse reaction
of HSDH. The data were fitted with a hyperbolic equation (wild type,
K0.5 = 60 ± 10 mM; Q443A,
K0.5 = 347 ± 16 mM; Q524A,
K0.5 = 335 ± 16 mM).
C, effect on the forward reaction of HSDH. The data were
fitted with a hyperbolic equation (wild type,
K0.5 = 12 ± 1 mM; Q443A,
K0.5 = 119 ± 6 mM; Q524A,
K0.5 = 92 ± 6 mM).
Kinetic constants for HSDH activities of the wild-type and mutant
AK-HSDH enzymes
Kinetic constants for AK activity of the wild-type and mutant
AK-HSDH enzymes
View larger version (18K):
[in a new window]
Fig. 4.
Native molecular mass determination of
wild-type and mutated AK-HSDH by chromatography on HiLoad 16/60
Superdex S 200 with 5 mM threonine (A) or
without effector (B). A 0.5-ml sample of
wild-type or mutant AK-HSDH (0.5 mg) was applied to the S200 column and
eluted at 1 ml/min in a buffer containing 50 mM Hepes-KOH,
150 mM KCl, 10% glycerol and without or with 5 mM threonine. Standards used (2 mg) were thyroglobulin (669 kDa), apoferritine (443 kDa), threonine deaminase (used only for
the calibration without threonine; 238 kDa), -amylase (200 kDa), and
alcohol dehydrogenase (150 kDa).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 5.
Proposed model for the inhibition of AK-HSDH
by threonine. Active catalytic domains of AK and HSDH are
represented with squares, whereas inhibited catalytic
domains are represented with triangles. Threonine binding on
subdomain 1 would induce in parallel 1) conformational modifications of
subdomain 2 and 2) conformational modifications of AK catalytic domain
leading to AK inhibition. Conformational modification of subdomain 2 would induce the binding of a second threonine leading to
conformational modifications of HSDH catalytic domain and HSDH
inhibition.
strand and the last loop of the
loop-
-loop-
-loop-
motif of each subdomain of AK-HSDH belong to
an effector-binding site. Additional mutations (I441A and I522A)
carried out in the end of the first
strand of each subdomain of
AK-HSDH disclose that Ile441 and Ile522 also
belong to the two effector-binding sites (results not shown).
-loop-
-loop-
motif found in AK-HSDH and TD is present in
other allosteric proteins, homology search was carried out using the
Pfam protein families data base (25).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Claude Alban, Dr. Michel Matringe, and Professor Roland Douce for helpful discussions.
![]() |
FOOTNOTES |
---|
* 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.
Present address: Laboratoire de Physiologie Cellulaire
Végétale, UMR 5019 CEA/CNRS/UJF, CEA-Grenoble, Avenue des
Martyrs, 38054 Grenoble cedex 9, France. E-mail: rdumas@cea.fr.
§ To whom correspondence should be addressed: Laboratoire de Physiologie Cellulaire Végétale, UMR 5019 CEA/CNRS/UJF, CEA-Grenoble, Avenue des Martyrs, 38054 Grenoble cedex 9, France.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M207379200
2 G. Curien, unpublished result.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: AK, aspartate kinase; TD, threonine deaminase, HSDH, homoserine dehydrogenase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Cohen, G. N. (1985) Methods Enzymol. 113, 596-599[Medline] [Order article via Infotrieve] |
2. | Viola, R. E. (2001) Acc. Chem. Res. 34, 339-349[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Zakin, M. M.,
Duchange, N.,
Ferrara, P.,
and Cohen, G. N.
(1983)
J. Biol. Chem.
258,
3028-3031 |
4. | Katinka, M., Cossart, P., Sibilli, L., Saint-Girons, I., Chalvignac, M. A., Le, Bras, G., Cohen, G. N., and Yaniv, M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5730-5733[Abstract] |
5. | Falcoz-Kelly, F., Janin, J., Saari, J. C., Véron, M., Truffa-Bachi, P., and Cohen, G. N. (1972) Eur. J. Biochem. 28, 507-519[Medline] [Order article via Infotrieve] |
6. | Bearer, C. F., and Neet, K. E. (1978) Biochemistry 17, 3512-3516[Medline] [Order article via Infotrieve] |
7. |
Fazel, A.,
Guillou, Y.,
and Cohen, G. N.
(1983)
J. Biol. Chem.
258,
13570-13574 |
8. | Fazel, A., Müller, K., Le, Bras, G., Garel, J.-R., Véron, M., and Cohen, G. (1983) Biochemistry 22, 158-165[Medline] [Order article via Infotrieve] |
9. | Ferrara, P., Duchange, N., Zakin, M., and Cohen, G. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3019-3023[Abstract] |
10. | Omori, K., Imai, Y., Suzuki, S.-I., and Komatsubara, S. (1993) J. Bacteriol. 175, 785-794[Abstract] |
11. | Omori, K., and Komatsubara, S. (1993) J. Bacteriol. 175, 959-965[Abstract] |
12. | Weiseman, J. M., and Matthews, B. F. (1993) Plant Mol. Biol. 22, 301-312[Medline] [Order article via Infotrieve] |
13. | Ghislain, M., Frankard, V., Vandenbassche, D., Mattews, B. F., and Jacobs, M. (1994) Plant Mol. Biol. 24, 835-851[Medline] [Order article via Infotrieve] |
14. |
Muehlbauer, G. J.,
Somers, D. A.,
Matthews, B. F.,
and Gengenbach, B. G.
(1994)
Plant Physiol.
106,
1303-1312 |
15. |
Gebhardt, J. S.,
Weisemann, J. M.,
and Matthews, B. J.
(1999)
Plant Physiol.
120,
339 |
16. | Paris, S., Wessel, P., and Dumas, R. (2002) Protein Expression Purif. 24, 105-110[CrossRef][Medline] [Order article via Infotrieve] |
17. | Gallagher, D. T., Gilliland, G. L., Xiai, G., Zondlo, J., Fisher, K. E., Chinchilla, D., and Eisenstein, E. (1998) Structure 6, 465-475[Medline] [Order article via Infotrieve] |
18. | Wessel, P., Graciet, E., Douce, R., and Dumas, R. (2000) Biochemistry 39, 15136-15143[CrossRef][Medline] [Order article via Infotrieve] |
19. | Bryan, P. A., Cawley, R. D., Brunner, C. E., and Bryan, J. K. (1970) Biochem. Biophys. Res. Commun 41, 1211-1217[Medline] [Order article via Infotrieve] |
20. | Paris, S., Wessel, P., and Dumas, R. (2002) Protein Expression Purif. 24, 99-104[CrossRef][Medline] [Order article via Infotrieve] |
21. | Chua, N. H. (1980) Methods Enzymol. 69, 434-436 |
22. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
23. | Scopes, R. K. (1974) Anal. Biochem. 59, 277-282[Medline] [Order article via Infotrieve] |
24. | Gourgeon, C., and Deléage, G. (1994) Protein Eng. 7, 157-164[Abstract] |
25. |
Bateman, A.,
Birney, E.,
Durbin, R.,
Eddy, S. R.,
Finn, R. D.,
and Sonnhammer, E. L. L.
(1999)
Nucleic Acids Res.
27,
260-262 |
26. | Chipman, D. M., and Shaanan, B. (2001) Curr. Opin. Struct. Biol. 11, 694-700[CrossRef][Medline] [Order article via Infotrieve] |