From the Department of Biological Sciences and
¶ Department of Horticulture and Landscape Architecture,
Purdue University, West Lafayette, Indiana 47906-1392
Received for publication, December 2, 2002, and in revised form, January 24, 2003
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ABSTRACT |
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The first step of proline biosynthesis is
catalyzed by Osmoregulation is of significance for agriculture because water is
a major limiting factor for crop productivity. Plants evolved a variety
of mechanisms for adapting to environmental stresses, one of which is
accumulation of proline under salinity, dehydration, or freezing
environments (1, 2). It has been proposed that proline plays an
important role as a compatible solute, maintaining proper balance
between the extracellular and intracellular osmolality. However, a
number of other functions have been hypothesized for this imino acid,
such as carbon or nitrogen reservoir, radical scavenger, or regulator
of intracellular pH (3, 4). Plants have two proline biosynthetic
pathways, the glutamate pathway and the ornithine pathway, with the
former appearing to play a predominant role under osmotic stress (5).
In the glutamate pathway, glutamate is converted by
GK1 to Genes specifying P5CS and P5C reductase have been cloned from plants,
making it feasible to test the role of proline under osmotic stress and
to aim to improve the salinity tolerance of agriculturally important
crops by genetic engineering. It has been reported that the first step
catalyzed by P5CS is the rate-limiting step in proline biosynthesis in
plants (7-9). Osmotic stress was shown to increase the P5CS mRNA
levels along with the proline pool size in Arabidopsis
thaliana and rice (Oryza sativa), suggesting that
transcriptional regulation of P5CS could be important for a control of
proline biosynthesis (10-12).
Tomato (Lycopersicon esculentum) has two distinct genes for
P5CS, called tomPRO1 and tomPRO2 (13). Although
this species accumulates more than 15-fold higher levels of proline
than A. thaliana or rice, the tomPRO1 and
tomPRO2 mRNAs are expressed essentially at constitutive
levels at low and high osmolality (13). These observations suggest that
some post-transcriptional control mechanism must be taken into account
for the regulation of proline synthesis in tomato. The GKs encoded by
the tomPRO1 locus of tomato, the P5CS genes of
Vigna aconitifolia, and grapevine (Vitis
vinifera) have been demonstrated to be subject to feedback regulation by proline (14-16). Roosens et al. (17)
described the isolation of a salt-tolerant mutant of Nicotiana
plumbaginifolia that lost proline feedback inhibition of the P5CS.
Also, Hong et al. (18) reported a direct role of feedback
inhibition of P5CS for proline accumulation by constructing transgenic
plants that expressed a mutated version of the V. aconitifolia P5CS, whose proline feedback regulation was lost.
These observations suggest that allosteric control of GK reaction also
plays a key role in regulating proline synthesis in plants (16,
18).
Studies with purified preparations revealed that the bacterial GKs and
the plant P5CS enzymes are subject to feedback regulation by proline at
very different sensitivities. For the bacterial GKs, the concentration
of proline resulting in 50% inhibition of activity (apparent
Ki) was in the range of 0.01-0.1 mM
(19-21), whereas for the plant P5CS enzymes, the apparent
Ki was estimated to be On the basis of primary sequence similarity, GKs have been assigned to
the so-called amino acid kinase family of enzymes (PF00696 in the Pfam
data base, www.sanger.ac.uk/Software/Pfam), which also includes AK, CK,
CK-CPS, NAGK, and uridylate kinase. Initial sequence comparisons
identified two sequence features that are common to GKs (see Fig. 1). A
phosphate binding site and a leucine zipper have been suggested in the
GKs, although neither seems to have a perfect match to the respective
consensus sequences (11, 14). The three-dimensional structures for CK,
CK-CPS, and NAGK are known (29-31), and the assignment of the
phosphate binding site has been supported by the sequence information.
Omori et al. (19) identified a motif common to GKs and AKs,
which they suggested to be part of the catalytic site. This motif
(Region II in Fig. 1), which can be recognized in other
members of the amino acid kinase family, has been corroborated
by the three-dimensional sequence information to contain part of the
nucleotide and aminoacyl binding sites.
To identify residues in GK that contribute to allosteric regulation and
to gain insights into the structure-function relationship of GK enzyme,
we carried out random mutagenesis of the tomPRO1 DNA to
alter the allosteric properties of the enzyme. The tomPRO1 clone, which has been isolated from a tomato cDNA library by
complementation of a proB (GK) mutation in E. coli, specifies GK and GPR as two separate polypeptides in two
non-overlapping open reading frames, similar to prokaryotic operons
(15). Phylogenetic analysis revealed that the GK and GPR polypeptides
encoded by the tomPRO1 locus are more similar to prokaryotic
enzymes than to eukaryotic counterparts, suggesting that GK may have
been incorporated into the tomato nuclear genome from a prokaryote by
horizontal gene transfer (13). Although unusual for eukaryotic
transcripts, a similar bi-cistronic organization has also been observed
in a locus specifying two subunits of another amino acid biosynthetic
enzyme, carbamoyl phosphate synthetase in alfalfa (33). The
tomPRO1 GK seems to be suitable for the analysis of the
determinants of allosteric control because it is shorter by ~100
amino acid residues in its C terminus than most other prokaryotic GKs.
Based on sequence comparisons, this ~100-amino acid tail has been
suggested to constitute an RNA binding domain (34), but the relevance
of this element to the activity or regulation of GK is unknown. The
tomPRO1 GK nevertheless contains the essentials for
catalysis and is subject to proline feedback inhibition (15). In this
work, we describe the identification and analysis of 13 different amino
acid substitutions at 9 different positions in tomPRO1 GK
that eliminated feedback regulation by proline, and we discuss the
possible roles of these amino acids in a predicted model structure of
GK.
Culture Media--
Minimal medium used was M63 (35) containing
10 mM glucose and 0.1 mM thiamine-HCl unless
otherwise stated. M63 was supplemented, as indicated, with a mixture of
19 amino acids consisting of 0.2 mM solutions each of the
protein amino acids except proline. In media containing 0.6 M NaCl, we used an 18-amino acid mixture that contained 0.2 mM each amino acid except proline and cysteine (because the
latter amino acid has been shown to be inhibitory in media of high
osmolality (36)). Complex medium was LB (37) supplemented with 100 µg/ml Ap as indicated. When used, the proline analog
3,4-dehydro-DL-proline (Dhp) (Sigma) was present at 1.5 mM.
Isolation of Dhp-resistant Mutants--
Basic techniques of DNA
manipulation were carried out according to the procedures described by
Sambrook et al. (38). Plasmid pPRO1 is a derivative
BluescriptKSII+ carrying the tomPRO1 locus (15). To
introduce mutations into the tomPRO1 GK, this plasmid was
transformed into the E. coli mutator strain XL-1 Red
(mutD mutS mutT) according to a supplier's instructions
(Stratagene, La Jolla, CA). Transformants were inoculated to LB plates
containing Ap and grown at 37 °C for 26-32 h. Plates containing the
transformant colonies were taken up and pooled in 2 ml of LB, and
plasmids were isolated from the pooled transformants with the Qiagen
plasmid purification procedure (Qiagen, Valencia, CA). This plasmid
preparation was transformed into the E. coli proline
auxotroph CSH26 ( GK/GPR-coupled Assays--
Derivatives of strain CSH26 carrying
the wild type or mutated pPRO1 plasmids were grown overnight in LB plus
Ap, and 0.1-ml samples were inoculated to LB Ap and grown overnight at
30 °C. The lawn of cells was taken up in 5 ml of LB, sedimented by
centrifugation, resuspended in 1 ml of 50 mM
HEPES.KOH (pH 7.2) containing 10 mM
dithiothreitol, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, and 100 µg/ml lysozyme. The crude extracts were centrifuged, and the supernatants were used to measure GK/GPR-coupled activity in 50 mM MOPS.KOH (pH
6.5), 8 mM MgCl2, 75 mM sodium
glutamate, 4 mM ATP, 0.4 mM NADPH, and various
concentrations of proline, as described by García-Ríos
et al. (15). The GK/GPR-coupled activity was determined as
the rate of NADPH consumption (decrease in
A340), and the enzyme specific activity
was expressed as nmol of NADPH consumed (min·mg of cell
protein) Determination of Total Proline Levels--
CSH26 derivatives
carrying wild type or mutant versions of pPRO1 were inoculated into
liquid M63 supplemented with 20 mM glucose, 0.2 mM thiamine, and 18 amino acids and grown at 30 °C to
A600 of 1.0. Lysozyme was added to 100 µg/ml,
and cell debris was removed by centrifugation. Culture media were
passed through 0.22-µm membrane filters (Millipore, Bedford, MA) and
mixed vigorously with a drop of CHCl3 to kill surviving
cells, and the CHCl3 was separated by centrifugation.
Proline content in the supernatant was measured by bioassay using
Salmonella typhimurium strain TL131 ( DNA and Amino Acid Sequence Analyses--
DNA sequences were
determined using automated fluorescent sequence (ALFexpress;
Amersham Biosciences) or by the DNA sequencing service of Iowa State
University. Nucleotide and predicted amino acid sequences were analyzed
with programs in the Genetic Computer group (GCG) package of the
University of Wisconsin. Amino acid sequence alignment was performed
using PILEUP program and manually adjusted.
Modeling of Tertiary Structure of tomPRO1 GK--
As there were
no three-dimensional structures available for GKs from various
organisms, we searched the Protein Data Bank for homologous proteins
with known three-dimensional structures. The amino acid sequence of
tomPRO1 GK was submitted to 3D-PSSM servers
(www.sbg.bio.ic.ac.uk/~3dpssm), and CK, CK-CPS, NAGK (Protein Data
Bank entries 1B7B, 1E19, and 1GS5 respectively) were identified with
the highest PSSM scores. Other modeling servers (GTOP,
spock.genes.nig.ac.jp/~genome/gtop-j.html; UCLA-DOE fold server,
fold.doe-mbi.ucla.edu; PredictProtein server,
www.embl-heidelberg.de/predictprotein; 3D-Jigsaw,
www.bmm.icnet.uk/servers/3djigsaw) were used to search modeling
templates to tomPRO1 GK and returned the same proteins as
templates. A predicted tertiary model of tomPRO1 GK, based on crystal structures of these enzymes, was built and drawn using program Insight II, Homology and Discover (Accelrys, Tokyo, Japan).
Isolation of Dhp-resistant Mutants--
Wild type E. coli is inhibited on minimal medium by the proline analog Dhp
(40). Because high level accumulation of proline antagonizes Dhp,
proline overproducing mutants can be recovered by selecting
DhpR derivatives on minimal media (36). Random mutations
were introduced into tomPRO1 GK by propagating pPRO1 in the
mutator strain XL-1 Red (mutD mutS mutT). A pool of
mutagenized pPRO1 plasmids was transformed into CSH26
(
The tomPRO1 gene contains a unique HpaI site
between codons 186 and 187 of the GPR open reading frame, downstream of
the GK sequence; it is possible to obtain the entire open reading frame specifying a functional GK without active GPR by deleting the sequences
3' to this HpaI site (data not shown). GPR-coding sequences were removed in this manner from 3 of the 32 mutant pPRO1 plasmids, and
the resultant plasmids were transformed into E. coli
proB mutant G13, which is deficient in GK but has GPR (41).
Each of these plasmids not only complemented the proB
mutation of strain G13 strain but also conferred DhpR (data
not shown), suggesting that the latter phenotype was due to mutation(s)
within the GK sequence of tomPRO1. These data were consistent with the previous observations that single amino acid mutations in GK or the GK domain of bifunctional P5CS could result in
proline analog resistance and loss of allosteric control (19, 22-28).
Sites of the Mutations in GK--
The nature and location of
mutations in the GK region of tomPRO1 from each of the 32 plasmids were determined by DNA sequence analysis. Because previous
mutations that conferred resistance to proline analogs have been
reported to lie in the N-terminal third of GK (23-25, 27, 28, 32),
initially we concentrated on determining the nucleotide sequence of the
N-terminal ~600 bases of the GK-coding region. Each plasmid had a
single base pair substitution in this region, resulting in an amino
acid replacement (Table I), except for
Dr214. The latter plasmid contained two substitutions: an (a Enzymatic Confirmation That the Mutations Diminish Proline Feedback
Inhibition--
To test whether DhpR resulted from a loss
of proline feedback regulation in the 13 mutants that were sequenced,
the GK/GPR-coupled activities of the wild type and mutant enzymes were
determined in cell-free extracts. Each of the mutant enzymes had lower
specific activities compared with the wild type in assays carried out
in the absence of proline (Table II). The
specific activities of the enzymes carrying mutations I79T, M94T,
D147G, E153A, E153G, E153K, L154S, and S159P were 20-66% that of the
wild type activity. The most drastic reduction in specific activity was
caused by the A62T, A62V, I149F, D162G, and D162N changes, suggesting
that the residues at positions 62, 149, and 162 may have overlapping functions in catalytic activity and allosteric regulation (see a
section of three-dimensional modeling below).
We also determined the effect of proline on the GK activity of the
mutant enzymes. The proline inhibition curve of the wild type enzyme
was similar to the one observed previously with the purified enzyme
(42). In contrast, all of 13 mutants exhibited decreased sensitivity to
feedback regulation. Fig. 2 shows the proline inhibition curves in nine representative mutants. The apparent
Ki values for proline of the wild type enzyme was
about 0.09 mM, and the apparent Ki of
mutants ranged from 1.9 to 310 mM (Table II), representing
a 20-3500-fold increase.
To verify that the DhpR phenotype indeed was due to
increased intracellular proline levels and to check whether the proline levels were correlated with the residual feedback regulation of GK, the
total amounts of proline (cellular plus excreted into the medium) were
measured in E. coli strains carrying the wild type or mutant
versions of pPRO1. As can be seen in Table II, proline levels in
mutants were 10-1000-fold greater than in the wild-type, indicating
that in all the mutants, the DhpR phenotype was the
consequence of proline overproduction. In general, there was a good
correlation between the degree of deficiency in proline feedback
regulation and the proline levels produced by the strains. The I79T,
I149F, and S159P substitutions, which caused the least disruption of
allosteric regulation (apparent Ki = 1.9, 20, and 19 mM, respectively), resulted in the lowest proline
production (10-25-fold increase over the wild type), and conversely,
the L154S and E153K substitutions, which caused the most severe loss of
allosteric control (apparent Ki = 90 and 310 mM), conferred the highest production of proline (~900-fold increase). This generalization, however, does not hold for
all of the mutants. The apparent Ki of the GKs
containing the A62V and M94T substitutions was comparable with that of
mutants containing the I79T, I149F, and S159P substitutions, but the
former two mutations resulted in a ~4-19-fold higher production of
proline compared with that caused by the latter three mutations. The
D147G substitution increased the apparent Ki to 180 mM but caused lower production of proline than did the
L154S substitution. Because it is possible that the mutations altered
not only the allosteric properties of GK but also its in
vivo stability or interaction with GPR, it will be necessary to
carry out assays with purified GK to fully characterize the effects of
the mutations on the enzyme.
Growth rates of mutants in the presence of Dhp were also determined.
All mutants showed 2-3-fold faster growth rate than wild type in the
presence of this anti-metabolite (Table II). The growth rate in the
presence of Dhp was well correlated with the proline levels; strains
expressing the GK with the I79T, I149F, and S159P substitutions, which
produced the lowest proline levels, also showed the lowest growth rate.
However, E153K and L154S, which exhibited the most extreme loss of
feedback inhibition and accumulated the highest levels of proline,
nevertheless grew more slowly than most of other mutants. Inconsistency
between proline levels and growth rates in the presence of Dhp might be
due to physiological effects arising from limitation for some important
intermediate or accumulation of a toxic metabolite arising from proline
overproduction. To test whether loss of feedback sensitivity or proline
overproduction might be accentuated by combining pairs of mutations, we
introduced the A62T mutation in combination with the D147G, E153K,
L154S, and D162G mutations. However, none of the combinations of double mutations A62T/D147G, A62T/E153K, A62T/L154S, and A62T/D162G resulted in further enhancement of proline overproduction that would be consistent with an even greater loss of allosteric feedback regulation (data not shown).
A Cluster of Allosteric Mutations in the Region Between Residues
147 and 162--
We generated at least 13 different amino acid changes
that diminished or eliminated the sensitivity of the tomPRO1
GK to feedback inhibition by proline. Fig.
3 shows the alignment of 21 GKs from various species in the region corresponding to positions 147 to 162 of
the tomPRO1 GK, where we obtained 6 substitutions giving rise to DhpR. The cluster of residues identified here might
be a part of the allosteric regulatory domain. In all sequences,
the residues at positions corresponding to 147 and 162 of
tomPRO1 are invariably aspartates. The fact that mutations
at both of these positions (D147G and D162G; Table I) diminished
sensitivity to feedback regulation but did not eliminate catalytic
activity suggests that these highly conserved aspartates might be
important for allosteric regulation. Two other mutations were I149F and
L154S. Positions corresponding to 149 and 154 show a characteristic
preference for Ile, Leu, Met, or Val in most GKs (except for the ProB
enzyme of Bacillus subtilis); mutation of these hydrophobic
amino acids resulted in loss of allosteric control. Two other targets
for our mutations were at residues 153 and 159, which are highly
variable among species. Conceivably, these amino acids could make
species-specific contributions to the feedback regulation that may be
dependent on the context of other residues or might be required for the overall folding of the enzyme.
The fact that we obtained three different amino acid replacements at
the highly variable position 153 (Table I) points out an intriguing
aspect of the relationship among GK enzymes. One of these mutations
changed the Glu to Ala. The identical substitution had been isolated at
the corresponding site of the GK of E. coli (position 143 in
that polypeptide) (25). This replacement resulted in an 80-100-fold
decreased sensitivity to proline feedback inhibition in both in
tomPRO1 GK and the E. coli enzyme, as judged by
an increase in their apparent Ki.
A second mutation at position 153 in tomPRO1 GK introduced
Lys, resulting in a change of charge from
B. subtilis has two GKs: ProB, which is constitutive, and
ProJ, which is induced by osmotic stress (43). This organism elevates its proline levels upon osmotic stress by increased de novo
synthesis. It has been hypothesized that the ProJ enzyme might be
insensitive to allosteric feedback inhibition by proline (43). With the exception of the B. subtilis ProJ enzyme and the
Corynebacterium glutamicum enzyme, all other
prokaryotic GKs have an acidic residue at position 153. The
B. subtilis ProJ enzyme contains a basic amino acid (Arg) at
this position, making it similar to the plant enzymes. However, the
possibility that the B. subtilis ProJ enzyme has atypical
allosteric properties among prokaryotic GKs has not yet been investigated.
A third mutation at position 153 was Glu Mutations in N-Terminal Third of GK--
Four mutations, A62T,
A62V, M94T, and I79T, which also decreased sensitivity to allosteric
feedback, are outside the region shown in Fig. 3. Position 62 is
occupied by an alanine in the GK/P5CS genes in 16 of 18 organisms.
Position 94 is not highly conserved but has a preference for a bulky,
hydrophobic amino acid (sequence comparison not shown). Finally, the
I79T mutation affected a residue that shows a high variability
(sequence comparison not shown). Like the highly variable resides at
positions 153 and 159, the Ile at position 79 might make a
species-specific contribution to the allosteric regulation of the
tomPRO1 GK or might be required for the proper conformation
of the enzyme.
Three-dimensional Modeling and Putative Proline Binding
Domain--
We were interested in seeing how the amino acid residues
that were identified to be involved in the allosteric regulation of GK
or P5CS might be positioned in a three-dimensional structure of the
protein. Although a crystallographic structure is not available for any
GK and P5CS, it is possible to make predictions about the tertiary
structure of GK by homology modeling programs based on the three
related enzymes, CK, CK-like CPS, and NAGK, whose structures have been
solved (29-31). CK catalyzes the phosphorylation of ADP by carbamoyl
phosphate during arginine catabolism, CK-CPS makes carbamoyl phosphate
by ATP-dependent phosphorylation of carbamate in the
pyrimidine and arginine biosynthetic pathways, and NAGK carries out the
phosphorylation of N-acetyl-L-glutamate by ATP
in the arginine pathway. Because different secondary structure prediction algorithms or threading methods (see "Experimental Procedures") recorded highly significant E-values using CK,
CK-CPS, and NAGK as the modeling template for tomPRO1 GK (E
values between e-14 and e-26 in 3D-PSSM program), the resultant
hypothetical model could provide useful insights into the structure and
regulation of this enzyme. However, it should be borne in mind that
with the current status of protein structure calculation, this
predicted model is highly speculative and may be dependent on the
modeling approaches used.
As might be expected from the similarities in the primary sequences of
CK, CK-CPS, and NAGK and in the reactions they catalyze, these enzymes
exhibit a high degree of structural resemblance. All three are
homodimers made up of subunits that have open
In addition to the 9 residues in tomPRO1 identified in this
study, 7 other residues corresponding to positions 115, 117, 118, 127, 150, 152, and 198 in tomPRO1 GK (Fig. 4), were shown to be important for allosteric regulation of GK or P5CS from other organisms. With the exception of residue 198, all of the other residues are located within the region that corresponds to the N-terminal domain of
the CK-CPS (residues 1-175 of tomPRO1 GK). The cluster of
mutations between 147 and 162 was localized to a sequence corresponding to the region that encompasses the interval between
The predicted allosteric domain in tomPRO1 GK appears to be
arranged to be able to make direct connection between the dimer interface and the substrate binding site. In fact, several residues within the regulatory domain can be assigned to residues of CK-CPS that
play a role in dimer formation (residues 118, 149, 150, 152, 153, 154 in tomPRO1 GK) or in substrate binding (residue 162 in tomPRO1 GK). Because the allosteric domain may be proximal
to the dimer interface and substrate binding site, a conformational alteration of the domain upon proline binding could affect catalytic activity or inter-subunit interaction. Residue 62 in tomPRO1
GK corresponds to a residue that functions as part of the phosphate binding site in CK-CPS and the N-acetylglutamate binding
site of NAGK, and residues 79, 94, and 127 were placed in
The exceptional residue 198 is in the putative C-terminal domain, which
has been suggested to be involved in nucleotide binding. Because it has
been reported that ATP and proline bind independently (20, 22) and
because the predicted ATP binding region is distant from the proline
binding motif in our proposed structure, the mutation at this position
may influence effector binding in an indirect manner. It may be noted
that the mutation at residue 198 actually caused a 4.5-fold increase in
the specific activity of the enzyme along with only a minor (~2-fold)
increase in the apparent Ki for proline (26).
Two conserved regions, I and II (Fig. 1), have been suggested to be
involved in phosphate and nucleotide binding in GKs, respectively (19).
Region I corresponds to sequences located in Conclusions--
Our data, obtained by genetic and biochemical
approaches and tertiary structure prediction, led to the identification
of residues important for allosteric regulation of GK and provide an
insight for the structure-functional relationship of this enzyme. The effector binding domain may mutually interact with or partly overlap components of the substrate binding sites and subunit interaction domain. Further detailed studies using a combination of various approaches including x-ray crystallography will be necessary to dissect
functional domains and residues essential for the catalytic and
regulatory properties of the enzyme. Knowledge gained from such studies
might allow us to control the flux of proline biosynthesis, which has
potential applications for development of proline-overproducing bacteria and engineering of improved adaptation to osmotic stress in
important crops (2).
-glutamyl kinase (GK). To better understand the
feedback inhibition properties of GK, we randomly mutagenized a plasmid
carrying tomato tomPRO1 cDNA, which encodes
proline-sensitive GK. A pool of mutagenized plasmids was transformed
into an Escherichia coli GK mutant, and proline-overproducing derivatives were selected on minimal medium containing the toxic proline analog 3,4-dehydro-DL-proline.
Thirty-two mutations that conferred 3,4-dehydro-DL-proline
resistance were obtained. Thirteen different single amino acid
substitutions were identified at nine different residues. The residues
were distributed throughout the N-terminal two-thirds of the
polypeptide, but 9 mutations affecting 6 residues were in a cluster of
16 residues. GK assays revealed that these amino acid substitutions
caused varying degrees of diminished sensitivity to proline feedback inhibition and also resulted in a range of increased proline
accumulation in vivo. GK belongs to a family of amino acid
kinases, and a predicted three-dimensional model of this enzyme was
constructed on the basis of the crystal structures of three related
kinases. In the model, residues that were identified as important for
allosteric control were located close to each other, suggesting that
they may contribute to the structure of a proline binding site. The putative allosteric binding site partially overlaps the dimerization and substrate binding domains, suggesting that the allosteric regulation of GK may involve a direct structural interaction between the proline binding site and the dimerization and catalytic domains.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-glutamyl
phosphate, which is reduced by GPR to
-glutamyl semialdehyde. This
product cyclizes spontaneously to P5C, which is reduced by NADPH to
proline by P5C reductase. In bacteria and yeast, GK and GPR reactions
are catalyzed by two separate enzymes. It has been shown in bacteria
that the GK activity is sensitive to feedback inhibition by proline
(6). In plants, the first two reactions of the pathway are mediated by
a bifunctional P5CS, consisting of a GK domain at the N terminus and a
GPR domain at the C terminus.
5 mM (16, 22).
Single amino acid substitutions have been first isolated in the GK of
Escherichia coli and Serratia marcescens, which
lessened or eliminated the sensitivity of the enzyme to allosteric
control. These mutations resulted in proline overproduction in whole
cells (23-25, 32), providing the most compelling proof that feedback
inhibition is indeed important for the regulation of proline synthesis
in vivo. In a site-directed mutagenesis of the V. aconitifolia P5CS, Zhang et al. (22) showed that amino
acid substitutions at positions 126 and 129 in the V. aconitifolia P5CS abolished proline feedback regulation. Other single amino acid mutations involving feedback regulation of GK have
been recently reported in Streptococcus thermophilus and Listeria monocytogenes (26, 27). In all, eight amino acid residues have been identified in six organisms as important for proline
feedback regulation of GK (22-24, 26-28, 32). However, because the
three-dimensional structure of GK has not been determined yet, there is
no corroborating information about roles of specific amino acids in the
allosteric binding site or other structure-function aspects of this protein.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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RESULTS AND DISCUSSION
REFERENCES
proBA), and transformants were selected
on LB Ap plates overnight. More than 2 × 104
transformant colonies were replicated to M63 + 19-amino acid plates
containing Dhp and incubated at 30 °C overnight. Approximately 400 colonies that were able to grow on Dhp-containing plates were streaked
again on the same medium. DhpR derivatives that grew faster
than CSH26 carrying wild type pPRO1 were tested on M63 + glucose + 18 amino acid plates containing 0.6 M NaCl as a further
step in identifying proline overproducing derivatives by virtue of
their increased salinity stress tolerance (36). Pilot experiments
indicated that increasing the concentration of Dhp above 1.5 mM did not change the frequency of DhpR,
suggesting that maximum Dhp uptake was accomplished at 1.5 mM. At this Dhp concentration, strain CSH26 carrying the
wild type pPRO1 did not grow at all. Re-transformation of the
mutagenized plasmids into an E. coli proline auxotroph
followed by subsequent screening for DhpR enabled us to
identify mutations in the plasmid-borne gene as opposed to chromosomal
loci, such as the putA or proP (proline transport) genes, where mutations could also result in DhpR
(6).
1.
proBA-47 putA842::Tn5). In this strain, in which
proline biosynthesis is blocked by the
proBA-47 mutation
and proline catabolism is blocked by the
putA842::Tn5 mutation, the biomass
yield is linearly dependent on the extracellular proline concentration
in the range of 0-200 µM (39). Strain TL131 was cultured
in M63 with 20 mM glucose, 18 amino acids, and 0.2 mM proline at 37 °C for 12 h, collected by
centrifugation, washed with the original volume of H2O to
minimize proline carryover, and resuspended at the original cell
density in M63 with 20 mM glucose. The suspension was
inoculated at 1:200 dilution into 2 ml of cell lysates of the
proline-overproducing strains to be tested, glucose was added to 20 mM, the cells were grown at 37 °C for ~24 h, and the
final cell yield was determined as the A600.
Because A600 is not proportional to the cell
density at A600 > 1, for those cultures that
reached this high density, the A600 was
determined on appropriate dilutions. A standard curve of the cell yield
as a function of proline concentration was generated with strain TL131,
grown under identical conditions in M63 containing 20 mM
glucose, 18 amino acids, and various input concentrations of proline.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
proBA), and derivatives carrying mutations in the
tomPRO1 gene that confer DhpR were obtained, as
described under "Experimental Procedures." Plasmid DNA was isolated
from 34 DhpR derivatives, transformed into strain CSH26,
selecting ApR, and re-tested for DhpR. Of the
34 plasmid lines, 32 proved to be able to confer DhpR in
the second round, confirming that this phenotype was caused by
mutations on the plasmid.
g) at
nucleotide position 763, which caused an Asp
Gly substitution at
amino acid position 162, and a (g
a) at nucleotide position 722, resulting in the change of a gtg (Val) codon to the synonymous gta
codon at amino acid position 148. Initial sequence analysis identified
13 different amino acid substitutions at 9 different residues. The
complete DNA sequence of a whole GK region for 15 clones (Dr246, -262, -161, -199, -220, -428, -122, -55, -319, -242, -385, -110, -63, -258, and -282), representing 13 different substitutions, was then
determined, confirming that they had only a single amino acid
substitution in the GK coding region that was caused by a single
nucleotide change, except for Dr214. Of the 13 mutations, 11 were g/c
a/t transitions, and the other two were a
t/c transversions.
Fig. 1 shows the locations of the nine
amino acid residues where replacements resulted in DhpR
phenotype. The identified amino acid residues were distributed throughout two-thirds of the N-terminus of GK, although a cluster was
readily apparent between positions 147 to 162, where 6 of the target
residues were concentrated. The facts that some of the mutations were
recovered multiple times and that some of the mutations resulted in
the substitution of different amino acids at the same residue (Table I)
suggest that we may have approached the saturation limit of mutations
generated in strain XL-1 Red, which resulted in a reduction in
sensitivity to allosteric regulation while retaining sufficient
catalytic activity for the synthesis of proline.
Location of Dhp-resistant mutations in tomPRO1 GK
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Fig. 1.
Positions in the primary structure of
tomPRO1 GK where mutations resulted in decreased
sensitivity to proline feedback inhibition. The positions
of nine amino acid are shown, where mutations resulted in
DhpR are indicated by red arrowheads. Region I
(blue) is the putative phosphate binding site and region II
(green), which has been originally identified as the
AK-homologous region (19), constitutes part of the nucleotide and acyl
substrate binding sites.
Characterization of mutant alleles of tomPRO1 GK
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Fig. 2.
Proline inhibition of wild type and mutant
form of tomPRO1 GK. GK/GPR-coupled assay was
carried out in crude extracts of proline auxotroph E. coli
strain harboring wild type (wt) or mutant versions of
tomPRO1. Results are expressed as percent of activities in
the absence of proline.
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Fig. 3.
Sequence alignment of proteins in GK family
from various organisms to the corresponding region of
tomPRO1 GK (positions 147-162). The
numbers are for the positions of the tomPRO1 GK.
The same or similar residues common to more than half of GKs are
shaded, and conserved positively charged residues in plant
P5CS enzymes at position 153 of tomPRO1 are
boxed. Triangles indicate residues where
amino acid substitutions obtained in tomPRO1 GK mutants
lacking of feedback regulation; open triangles show
positions that are occupied by an identical or similar amino acid in
all species, and closed triangles point out divergent
residues. Accession numbers for GK genes are B. subtilis
ProJ, F69682; B. subtilis ProB, P39820; C. glutamicum, U31230; E. coli, P07005; Hemophilus
influenzae, P43763; L. esculentum tomPRO1, U27454;
S. marcescens, P17856; Saccharomyces cerevisiae,
P32264; S. thermophilus GK, X92418: Synechocystis
sp., D90903; Thermus thermophilus, D29973;
Treponema pallidum, U61535. Accession numbers for P5CS genes
are: A. thaliana P5CSA, D32138; A. thaliana
P5CSB, X86778; Actinidia deliciosa, U92286;
Caenorhabditis elegans, Z50797; Homo sapiens,
X94453; L. esculentum tomPRO2, U60267; Medicago
sativa, X98421; O. sativa, D49714; V. aconitifolia, M92276.
1 to +1. GK sequences of
the plant P5CS enzymes invariably contain a positively charged amino
acid (Lys or Arg) at this position, whereas most of the bacterial GKs
have negatively charged residues (Asp or Glu) here (Fig. 3). The
presence of a Glu at this site in the tomPRO1 GK is
consistent with our classification of that protein as being more
similar to the prokaryotic type GKs than to the eukaryotic P5CS enzymes
(13). Interestingly, the E153K substitution caused the most marked loss
of feedback inhibition in tomPRO1 GK, as indicated by the
highest value of apparent Ki (Table II). The
~50-fold lower affinity of the plant P5CS enzymes for proline as an
inhibitor compared with that of the prokaryotic type GKs (please see
the Introduction) might be due to the positively charged amino acid at
the site corresponding to position 153 in the tomPRO1 GK. It
is possible that during evolution, the plant P5CS enzymes acquired and
fixed a residue at this position that diminished their sensitivity to
allosteric control as compared with the primordial prokaryotic type
GKs, resulting in a higher capacity for the accumulation of proline in
plants during osmotic stress.
Gly (Table I). It is
noteworthy that C. glutamicum already carries a Gly at the corresponding position (Fig. 3), but there is no information concerning the allosteric regulation of the enzyme from this organism.
/
structures
composed of 16
strands and 8
helices that are nucleated by a
central
sheet of 8 main strands sandwiched between two layers of
helices. Each subunit can be divided into N- and C-terminal domains
split by a large crevice. The N-terminal domain contains the aminoacyl
substrate binding site and entire dimer interface, the C-terminal
portion has the nucleotide binding site, and the crevice constitutes
the site for phosphate transfer from nucleotide to the carboxyl group.
As shown in Fig. 4, the threading model predicted a structure made up of eight strands of the main
sheet surrounded by
helices in tomPRO1 GK, similar to those
seen in CK-CPS. The conservation of eight strands of the main
sheet and the surrounding
helices is consistent with a proposal that the
amino acid kinase family of enzymes share this basic
characteristic (31). Therefore, it is likely that tomPRO1 GK
catalyzes phosphoryl group transfer with similar aminoacyl and ATP
binding domains as CK-CPS. GK may form dimers with similar interfacial
elements as the other three enzymes, although there are conflicting
reports as to whether GK forms dimeric or hexameric structures in
different organisms (20, 44).
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Fig. 4.
Comparison of the hypothetical secondary
structure of tomPRO1 GK with CK-CPS.
Arrows and cylinders above and below
the sequences depict strands and
helices, respectively. Eight
of main
strands are colored blue, and a small
sheet
is in purple. All other strands and helix D included in a
peripheral domain of CK-CPS are colored orange. Main helices
that make up the inter-subunit interface are in yellow, and
the remaining helices are in green. Red triangles
and red circles show important residues for feedback
regulation identified in this and other studies, respectively. Region I
of tomPRO1 GK, the putative phosphate binding site, and
region II, which shows high conservation to amino acid kinase family,
are highlighted by blue and green boxes,
respectively.
9-
10 and the
interval between
11-
F of CK-CPS, which consists of a large loop
containing hairpin
motif of
10 and
11. The three mutations that are between positions 115-118 in tomPRO1 GK can be
mapped to the region that includes the
4-
5 interval and
5 of
CK-CPS. This region, which exhibits different secondary structure
between CK-CPS and NAGK, corresponds in CK-CPS to the junction between a hairpin
motif of
4 and
8 and the peripheral domain and in NAGK to part of a hairpin
motif of
6-
7. Residues 115-118 and 147-162, where most of the mutations are concentrated, are folded very
close to each other within our putative three-dimensional structure of
the GK (Fig. 5), suggesting that they may
constitute part of the proline binding site. This putative allosteric
regulatory domain corresponds to a region of a small sheet consisting
of the two sets of antiparallel strands (
11-
10 and
4-
8 in
CK-CPS,
9-
10 and
7-
6 in NAGK) and a junction to a
peripheral protruding domain in CK-CPS and a loop in NAGK that is
emerging from the sheet. This region shows one of the major differences
among the enzymes of the amino acid kinase family. The variation in the structures was interpreted to reflect differences of binding of the
various substrates (31). We propose that the structural differences in
this domain in GK as compared with other family members might also
reflect the diversity in the regulatory properties of the enzymes. The
E. coli NAGK is not an allosteric enzyme (although the
orthologous enzyme from Pseudomonas aeruginosa and other
bacteria is subject to feedback regulation) (45), and there are no
reports on the feedback regulation of CK or CK-CPS. Thus, when the
structure of GK is available, it will be interesting to compare the
features that determine the allosteric properties of these related
enzymes.
View larger version (25K):
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Fig. 5.
A proposed three-dimensional model of
tomPRO1 GK. A, view from the upper
side of the molecule. B, view from the dimerization surface.
Nine amino acid residues that were identified in this study, and seven
residues that were identified by others are highlighted in
red and purple, respectively. Regions I and II
are shown in blue and green, respectively.
Helices D and
E in tomPRO1 GK, which correspond to
helices that are important for inter-subunit interaction in CK-CPS, are
colored yellow. The domain where mutations were closely
positioned may constitute a structure involved in allosteric regulation
by proline. The discontinuities in the ribbons correspond to regions of
tomPRO1 GK, where we were not able to calculate significant
conformational conservation against the template structure.
helices that are essential for dimerization in CK-CPS and NAGK. These results
also support the possibility of reciprocal communication among
substrate binding, subunit interaction, and allosteric regulation. Residues 62 and 162, where mutations resulted in the greatest decrease
in specific activity, are located at the borderline between the
allosteric domain and the catalytic domain in our predicted three-dimensional model, consistent with the possibility that these
residues could have an overlapping role in feedback regulation and
catalytic activity. The suggestion that there is an interaction between
the catalytic and allosteric domains is consistent with previous
biochemical analyses indicating that the binding of the proline could
affect the substrate binding (22). The observation that a mutation in
the proline binding site was found to confer decreased stability of GK
(21) is consistent with the suggestion that there is an interaction
between the proline binding site and other parts of the molecule that
determine subunit interaction, as suggested by our model.
B of CK-CPS, and region
II corresponds to sequences contained in structures
F to
12b in
CK-CPS (Fig. 4). The latter contains the loop that forms most of the
ATP binding site (
12-
13 loop in CK-CPS). These structural
elements for domains I and II are well conserved within the amino acid
kinase family, suggesting that they constitute an important structure
for ATP binding and phosphate transfer. The fact that these two
conserved regions were arranged within a central core rather than two
distant loops or unstructured domains corroborates the relevancy of our model.
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ACKNOWLEDGEMENTS |
---|
We thank E. Bremer for bringing to our attention the existence of the proline-insensitive GK in B. subtilis, D. Eisenberg for pointing out the similarities in the structure of GK and CK, H. Hatanaka for helpful insights on the three-dimensional modeling of tomPRO1 GK, D. Sanders for helpful discussions, and T. Smith for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the United States Department of Agriculture Grant 93-37100-8871.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: National Institute for Basic Biology, 38 Nishigounaka, Myo-daiji-cho, Okazaki 444-8585, Japan.
Present Address: National Center for Alternative Energy,
Environment, and Innovative Technology, C. R. Trisaia, S. S. Jonica Km-419-500, Rotondella (MT), Italy.
** Present address: Dept. of Natural Sciences, Texas A&M International University, Laredo TX 7804.
To whom correspondence should be addressed. Tel.: 765-494-4969;
Fax: 765-496-1496; E-mail: lcsonka@bilbo.bio.purdue.edu.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M212177200
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ABBREVIATIONS |
---|
The abbreviations used are:
GK, -glutamyl
kinase;
Ap, ampicillin;
AK, aspartate kinase;
CK, carbamate kinase;
CK-CPS, CK-like carbamoyl phosphate synthetase;
Dhp, 3,4-dehydro-DL-proline;
GPR,
-glutamyl phosphate
reductase;
LB, Luria-Bertani medium;
M63, minimal medium 63;
A600, optical density at 600 nm;
NAGK, N-acetyl-L-glutamate kinase;
P5C,
1-pyrroline-5-carboxylate;
P5CS, P5C synthetase
(bifunctional GK-GPR enzyme);
R, resistance to an
antibiotic or anti-metabolite;
MOPS, 4-morpholinepropanesulfonic
acid.
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