Download Cloning and expression of maize-leaf pyruvate, Pi dikinase

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Biochemical and Biophysical Research Communications 345 (2006) 675–680
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Cloning and expression of maize-leaf pyruvate, Pi dikinase
regulatory protein gene
Jim N. Burnell
a
a,*
, Chris J. Chastain
b
Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Qld 4811, Australia
b
Department of Biosciences, Minnesota State University-Moorhead, Moorhead, MN 56563, USA
Received 5 April 2006
Available online 4 May 2006
Abstract
Pyruvate, orthophosphate dikinase (PPDK; E.C. 2.7.9.1) catalyzes the synthesis of the primary inorganic carbon acceptor, phosphoenolpyruvate in the C4 photosynthetic pathway and is reversibly regulated by light. PPDK regulatory protein (RP), a bifunctional serine/
threonine kinase-phosphatase, catalyzes both the ADP-dependent inactivation and the Pi-dependent activation of PPDK. Attempts to
clone the RP have to date proven unsuccessful. A bioinformatics approach was taken to identify the nucleotide and amino acid sequence
of the protein. Based on previously established characteristics including molecular mass, known inter- and intracellular location, functionality, and low level of expression, available databases were interrogated to ultimately identify a single candidate gene. In this paper,
we describe the nucleotide and deduced amino acid sequence of this gene and establish its identity as maize PPDK RP by in vitro analysis
of its catalytic properties via the cloning and expression of the recombinant protein.
Crown Copyright 2006 Published by Elsevier Inc. All rights reserved.
Keywords: PPDK; PPDK regulatory protein; RP; C4 photosynthesis; Protein phosphorylation/dephosphorylation; Maize; Zea mays
Pyruvate, orthophosphate dikinase (PPDK), an enzyme
found in plants and certain microorganisms, catalyzes the
reversible phosphorylation of pyruvate to phosphoenolpyruvate (PEP) [1]:
ADP
AMP
RP
PPDK-Thr
PPDK-ThrP
PPDK
Pyr þ ATP þ Pi $ PEP þ AMP þ PPi
In C4 plants it serves to regenerate the primary CO2
acceptor PEP [1], while its role in C3 plants is not fully
understood [2]. As one of two potentially rate-limiting
enzymes of the C4 pathway [1,3], PPDK activity is strictly
light regulated post-translationally by reversible phosphorylation of an active-site Thr residue (Thr456 in maize). A
single, bifunctional protein kinase/phosphatase named
the PPDK regulatory protein (RP) catalyzes this regulatory
phosphorylation/dephosphorylation cycle [4,5]:
*
Corresponding author. Fax: +61 7 47816078.
E-mail address: [email protected] (J.N. Burnell).
Active
Inactive
PPi
Pi
It is a highly unusual regulatory enzyme in at least three
important respects. First, it is bifunctional in that it catalyzes both PPDK inactivation (phosphorylation) and activation (dephosphorylation). This is quite rare because most
regulatory phosphorylation cycles have separate kinase
and phosphatase enzymes [6,7]. Second, it uses ADP
instead of ATP as the phosphoryl donor. Third, it employs
a Pi-dependent, inorganic pyrophosphate (PPi)-forming
phosphorolytic dephosphorylation mechanism, as opposed
to simple hydrolysis as in most protein phosphatases [8].
0006-291X/$ - see front matter Crown Copyright 2006 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2006.04.150
676
J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680
Although the protein has been partially purified a number of times (e.g., see [4,8,9]) the enzyme has defied all
attempts to determine its molecular sequence. Specifically,
the enzyme is of such low abundance in the final fractions
that obtaining authentic peptide sequences from onedimensional SDS–PAGE gels has proved elusive. In what
appeared to be a breakthrough for its cloning, Burnell,
using a similar purification protocol, was able to obtain a
preparation of RP polypeptide that generated antibodies
capable of inhibiting the RP inactivation reaction in assays
containing chloroplasts as a source of both RP and PPDK
(unpublished results). When these antibodies were used to
screen a maize leaf cDNA expression library for candidate
RP clones, an RP-encoding clone could not be recovered.
Thus, without authentic peptide sequence or highly specific
antibodies, cloning of RP using conventional approaches
was essentially precluded. More recently, advanced
approaches for cloning RP, such as the bacterial two-hybrid system, were attempted with PPDK serving as the bait
protein in a bacterial cell line transformed with a maize leaf
cDNA expression library. However, thorough screening of
two-hybrid libraries consistently failed to identify clones
that encoded proteins capable of catalyzing ADP-dependent inactivation of PPDK. Nor did the use of site-directed
mutagenesis of the bait PPDK regulatory threonine and/or
the catalytic-histidine residues (e.g., in attempts to increase
the interaction or dwell time of RP with PPDK) reveal a
positive clone (Tems and Burnell, unpublished results).
It was with the advent of ever-expanding database
sequences for a range of vascular plant species that we finally
opted for a bioinformatics approach as the most viable
means for cloning the RP gene. We sought to do this by
identifying those sequences that were consistent with the
known properties of the RP. These included plant proteins
with (i) an estimated molecular mass of between 45 and
48 kDa, (ii) a chloroplast transit peptide, (iii) a threonine/
serine protein kinase domain, (iv) a phosphate/pyrophosphate binding domain, and (v) a relatively high proportion
of hydrophobic residues (as based on its salting-out profile).
Moreover, we expected the gene to be classified as having
‘‘unknown function’’ based on the absence of any other
eukaryotic protein of like catalytic function. Additionally,
since RP activity has now been demonstrated in C3 plant
chloroplasts [2] we also expected C3 plant RP homologs to
be present in C3 plant databases. From these properties, a
number of likely candidates were identified in maize and then
cross-referenced with the rice and Arabidopsis genome databases. Augmenting our database search was a recently published proteomics study that compared expression levels of
soluble, stromal polypeptides in isolated maize-leaf bundle
sheath and mesophyll cell chloroplasts [10]. This information facilitated the identification of the most likely RP gene
candidate within our search (GenBank Accession Nos.
AY106112 and AY106855). We then generated a PCR product corresponding to the respective ORF sequence of this
gene and subsequently utilized it as a probe for screening a
maize-leaf cDNA library. Positive clones were recovered as
excised plasmids and the cDNA insert subsequently
subcloned into an Escherichia coli expression vector for
producing the encoded protein recombinantly. Reported
here are experiments performed with this recombinant protein that confirm its identity as the maize PPDK bifunctional
regulatory protein.
Materials and methods
A maize leaf cDNA library constructed in kZAP (Stratagene) was
screened with a radioactively labeled 491 bp PCR fragment generated
using the cDNA library as template and two internal primers identified
using MacVector. Sequences of the two primers were 5 0 CCACCTCTTCTCCTTGATTGACG-3 0 (forward primer) and 5 0 CAGTCCAAAAACCTTGTCCTGG-3 0 (reverse primer). About 25 ng
DNA was radiolabeled using a Decaprime Labeling Kit (Ambion) and
[a-32P]dATP. Positive plaques were isolated and rescreened and pure
plaques isolated. Plasmid DNA was obtained by in vivo excision using
Helper phage according to the manufacturer’s instructions (Stratagene).
The nucleotide sequence of the plasmid DNA was determined (Macrogen,
Korea).
An insert containing the coding region of full length DNA was obtained
by digesting plasmid DNA with NheI, a restriction enzyme that digests the
plasmid insert six bases upstream of the initiating ATG start codon, and
KpnI which digests within the polylinker of pBluescript, and ligated into
pROExC digested with SpeI and KpnI. Following heat transformation of
competent NM522 cells in the presence of ligated DNA, cells were grown
overnight on LB carbenicillin (LBC) plates and colonies used to inoculate
5 ml LBC cultures. After overnight shaking a sample of each culture was
removed and used to prepare a glycerol stock. Plasmid DNA was isolated
and digested with a variety of restriction enzymes to confirm the presence of
the insert in the vector. Glycerol stocks of cultures containing cells with
successfully cloned inserts were used to inoculate 5 ml cultures which were
then used to inoculate 500 ml cultures (in 2 L baffled flasks) and shaken.
After 3 h IPTG was added to a final concentration of 0.5 mM and the
cultures grown overnight at 25 C. Cells were harvested by centrifugation,
washed with extraction buffer containing 25 mM Tris–HCl, pH 7.5, 5 mM
MgCl2, 1 mM Pi, and 0.5 mM b-mercaptoethanol, the cells resuspended in
5 ml of buffer and frozen at 80 C. Cells were sonicated (10 s bursts for
2 min at 80% output—Branson Sonifier 450) and the cell debris removed by
centrifugation at 40,000g for 30 min. The crude supernatant was decanted
and applied to a 5 ml nickel–NTA affinity column (Pharmacia) and washed
with buffer until the OD280nm decreased below 0.5. The column was then
washed sequentially with buffer containing 5 mM imidazole until the
OD280nm decreased below 0.5 and protein eluted from the column with a
solution containing 20 mM imidazole in buffer. Those fractions containing
eluted protein were tested for ADP-dependent PPDK inactivation activity
and Pi-dependent activation.
Inactivation assay. RP catalyzed inactivation of active (dephospho)
maize C4 recombinant PPDK (EC 2.7.9.1; GenBank Accession No.
J03901) was performed using a previously published procedure including
the following modifications [8]. Pre-activated (pre-warmed at 25 C for
60 min) Ni–NTA purified recombinant maize C4 PPDK [11] (0.15 U) was
added to a reaction mixture containing 0.1 ml column buffer and 25 ll of
recombinant RP containing column eluate in a final volume of 0.2 ml.
Inactivation reactions were initiated by the addition of 20 ll of 20 mM
ADP/1 mM ATP. At various intervals, 20 ll samples were withdrawn and
assayed for PPDK activity using a spectrophotometric assay as previously
described [12]. After 10 min incubation, 50 ll of the inactivation assay
reaction was added to an equal volume of SDS–PAGE buffer (125 mM
Tris–HCl, pH 6.8, 12% w/v SDS, 10% v/v glycerol, 0.01% w/v bromophenol blue, and 0.5% b-mercaptoethanol). The quenched samples were
then heated at 95 C for 5 min prior to loading aliquots on SDS–PAGE
gels as described below.
Pi-dependent PPDK reactivation assays. Purified recombinant PPDK
and RP were incubated with 2 mM ADP/0.1 mM ATP at 25 C. After
J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680
10 min the whole incubation mixture was passed through a column
(0.4 · 6 cm) of Sephadex G25 to remove the nucleotides [5]. Inorganic
phosphate was added to the column eluate to a final concentration of
1 mM and 25 ll samples removed and spectrophotometrically assayed for
PPDK activity [12].
SDS–PAGE and Western blot analysis. Proteins present in the terminated reaction mixtures were separated by SDS–PAGE on 10% polyacrylamide gels [13] and the separated proteins transferred to HybondP
(Amersham) membrane. The resulting blots were probed with either one
of two primary maize PPDK rabbit polyclonal antibodies. For specific
detection of RP catalyzed Thr456-phosphorylated PPDK, affinity-purified
polyclonal antibodies raised against a synthetic phosphopeptide conjugate
corresponding to the phosphorylation domain of maize C4 PPDK
(encompassing residues 445–464 [AVGILTERGGMpTSHAAVVAR]
[14,15]) were used. For detection of total PPDK (phospho- and dephospho-) polyclonal antibodies raised against the recombinant maize C4
PPDK monomer were used. For following His-tagged recombinant RP
during purification, anti-Tetra-His antibodies (Qiagen) were also used.
Proteins on Western blots were visualized using a secondary antibody
conjugated to either horseradish peroxidase or alkaline phosphatase.
Results and discussion
Identification of a potential RP candidate using in silico
bioinformatics screening
Previous in vitro studies of partially purified maize leaf
RP demonstrated that RP was functionally a threonine/
serine kinase in that Ser but not Tyr could substitute for
its target Thr456 residue [2,14,15] yet utilizing ADP as
the phosphoryl donor [8]. In previous reports where the
protein had been purified [4,8,9] the protein was shown
to precipitate at low ammonium sulfate concentrations
suggesting the protein is relatively hydrophobic. Furthermore, maize RP has also been demonstrated to be co-localized along with its target protein, PPDK, to the chloroplast
stroma of C4 leaf mesophyll cells [16]. Thus, along with
PPDK as a nuclear encoded protein, its gene must also
encode a chloroplast transit peptide [17]. Additional
insights into RP gained from previous purification studies
include an estimated molecular mass of 45–48 kDa and
that it is typically present in maize leaves as a very low
abundant protein [8,9]. Lastly, since no other known
eukaryotic protein possesses the bifunctional catalytic
properties of RP, we expected any RP candidate genes to
be classified as ‘‘unknown’’.
These collective characteristics were then utilized for
interrogating the maize databases for candidate RP genes.
From this search, a list in excess of about 50 maize proteins
was compiled and these sequences cross-referenced with
related sequences in the rice and Arabidopsis databases.
As this search was nearing completion, a comparative proteomics study was published that profiled differences in soluble chloroplast polypeptide content between maize leaf
bundle sheath and mesophyll cell-types [10]. We noted that
a partial peptide sequence corresponding to an unknown
protein [ZmGI Accession No. TC220929] was of apparent
low abundance and expressed more or less exclusively in
mesophyll chloroplasts (bundle sheath: mesophyll ratio of
0.04). More extensive sequence information indicated the
677
corresponding pre-protein had a molecular mass of
45.9 kDa including a predicted (ChloroP) [18] chloroplast
transit peptide of 42 amino acids, leaving a mature protein
with a molecular mass of 41.6 kDa.
We then proceeded to clone the corresponding cDNA
for the ZmGI TC220929 gene (GenBank Accession No.
AY106855) by screening a maize leaf cDNA library with
a cloned 491 bp PCR fragment complementary to sequences internal to the respective AY106855 ORF. Screening of
the library with this 491 bp 32P-labelled probe detected
about 50 positive plaques out of 25,000 plaques. In vivo
excision of plasmid DNA from the lambda clones produced ‘‘two families’’ of clones that differed in sequence
length within the 3 0 -non-coding region (as confirmed by
DNA sequencing). Examination of the amino acid
sequences of these two maize leaf RP clones with SignalP
3.0 [19] and ChloroP 1.1 [18] suggested a common chloroplast transit sequence with a cleavage site between amino
acid residues 42 and 43, leaving a mature protein of 384
amino acid residues with a molecular mass of about
41.5 kDa. A more detailed examination of the full-length
protein with PROSITE [20] identified (i) a protein kinase
C phosphorylation site, and (ii) an ATP/GTP-binding
motif A (P-loop) between residues 293 and 300, both consistent with the functional identity of the protein as a serine/threonine protein kinase. Interestingly, a protein
BLAST [21] search across all phyla revealed that this protein shares a large, centrally oriented conserved domain
called DUF299 (Domain of Unknown Function) with a
relatively large number of PPDK-possessing bacterial species, in addition to rice and Arabidopsis.
For ascertaining the identity of this gene as RP, we
needed to show in vitro that the recombinantly produced
protein had the unique catalytic properties of maize leaf
RP. That is, Thr456-specific, ADP-dependent phosphorylation of the PPDK catalytic intermediate (PPDKHis458-P) and Pi-dependent dephosphorylation of the
same ThrP residue. To accomplish this, the ORF of the
shorter clone was subcloned into an E. coli expression
vector that encoded a 6· His-tag N-terminal to the translational start codon of the native ORF. The expressed
pre-protein was purified to >80% purity by Ni-affinity
column chromatography (Fig. 1, lane 4). This RP active
fraction was eluted with 20 mM imidazole with an apparent molecular mass of 40 kDa. This preparation was used
in subsequent ADP-dependent PPDK inactivation assays
(Table 1) and for the Western blot-based in vitro phosphorylation/dephosphorylation assays (Fig. 2B).
In the in vitro test of the recombinant protein to inactivate PPDK in an ADP-dependent manner, we clearly show
robust inactivation of active, recombinant maize C4 PPDK
(Table 1). Importantly, not only was the inactivation
shown to be ADP-dependent, but that pyruvate could
inhibit PPDK inactivation by this protein further underscores its identity as RP. This specificity is inferred because
only the catalytic intermediate form of PPDK (phosphoHis458) can serve as the phosphorylation substrate of
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J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680
MW 1
kDa
110
77
2
3
4
5
6
A MW 1
2
3
4
5
6
44
29
B MW
Fig. 1. Coomassie blue-stained SDS–PAGE of bacterially expressed RP
purified by nickel-affinity column chromatography. RP, expressed in 1 L
of bacterial culture, was purified on a 5 ml nickel affinity column. The
column was loaded with crude extract (lane 2), the column washed with
column buffer (lane 1), 5 mM imidazole in column buffer (lane 3), 20 mM
imidazole in column buffer (lane 4), and 200 mM imidazole in column
buffer (lane 5). Purified maize recombinant PPDK was loaded in lane 6.
MW; prestained molecular weight markers.
RP, not simply the enzyme with an unphosphorylated
Thr456 residue [14,22,23]. Thus, by inclusion of the substrate pyruvate in the inactivation reaction the His458
phosphate is immediately transferred to pyruvate catalytically (in forming PEP) thus converting PPDK back to
the non-phosphorylated, non-RP substrate His458 state
(Table 1 and Fig. 2). Western blots of quenched aliquots
of the PPDK inactivation reactions probed with maize C4
PPDK phosphopeptide antibodies provided unequivocal
immunological confirmation that the recombinant protein
catalyzed site-specific (Thr456) phosphorylation of PPDK.
A companion blot probed with maize C4 PPDK
(phospho + dephospho) antibodies indicated the presence
of equal amounts of maize C4 recombinant PPDK in each
of the reaction mixtures, indicating the phospho-PPDK
signals in Fig. 2B can be viewed as quantitative
(Fig. 2A). For demonstrating that the cloned recombinant
protein could also catalyze the opposing RP reaction, that
is, Pi-dependent dephosphorylation of Thr456-P-PPDK,
the end-point reaction mixture from the PPDK inactivation/phosphorylation assay was desalted by Sephadex G25 column chromatography and re-incubated for 10 min
in the presence of 1 mM Pi. Aliquots of this reaction were
1
2
3
4
5
6
Fig. 2. Inactivation of PPDK by bacterially expressed RP. Inactivation
reactions were run and terminated reaction mixtures subjected to SDS–
PAGE, proteins transferred to Hybond-P membrane, and proteins located
using. (A) Rabbit anti-PPDK antibodies. (B) Rabbit anti- Thr456-P
phosphopeptide antibodies. Lane 1, complete reaction; lane 2, minus
ADP/ATP; lane 3, minus RP; lane 4, PPDK inactivated in a complete
reaction, the reaction mixture run through a Sephadex-G25 column, and
activation continued for 10 min with 1 mM Pi; lane 5, no PPDK; lane 6,
complete reaction plus 2 mM pyruvate.
analyzed for phospho-PPDK content via Western blots
probed with maize C4 PPDK phosphopeptide antibody
as before. As evident from the signal intensity of the immunoreactive bands in lanes 1 and 4, Fig. 2B, the recombinant
RP was able to catalyze a decrease in the level of phosphoPPDK (versus control). This Pi-dependent decrease in the
amount of phosphorylated (inactivated) PPDK is consistent with the corresponding level of increase in PPDK
activity as determined in a parallel PPDK spectrophotometric-based activation assay of the same desalted reaction
mixture (64% of the original PPDK activity, Table 1). Further confirmation that the recombinant protein possessed
RP-like, Pi-dependent PPDK activation property was provided by the demonstration that adding inorganic pyrophosphatase to the PPDK activation reaction
significantly increased the rate and final extent of activation
Table 1
ADP/ATP-dependent inactivation of purified PPDK by bacterially-expressed RP
Reaction no.
Reaction mixtures
PPDK activity at time zero (%)
PPDK activity after 5 min (%)
PPDK activity after 10 min (%)
1
2
3
4a
5
6
7
Complete
Minus ADP/ATP
Minus RP
Complete
Plus 2 mM pyruvate
Minus PPDK
Plus 2 mM pyruvate
100
100
100
Not assayed
100
0
100
70
104
93
Not assayed
97
0
97
47
108
105
Not assayed
99
0
99
PPDK and ADP-dependent inactivation activities were assayed as described in Materials and methods.
a
PPDK activity of reaction 4 was not determined during inactivation. Following the addition of ADP/ATP, reaction 4 was incubated at 25 C for
10 min and then subjected to Sephadex G25 column chromatography. The desalted reaction mixture was then incubated at 25 C for 10 min with 1 mM Pi
and PPDK activity determined to be 64% of the time zero activity of reaction 1. Remaining reaction mixtures were subjected to SDS–PAGE and Western
blot analysis (Fig. 2).
J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680
of inactivated PPDK (result not shown). This is consistent
with the RP dephosphorylation mechanism as a Pi-dependent PPi forming phosphorolytic cleavage, as opposed to a
simple hydrolysis as with most protein phosphatases. Thus,
removal of PPi by pyrophosphatase should thermodynamically ‘‘pull’’ the reaction forward [4], as is demonstrated
here.
As mentioned above, PPDK is not only present in plants
but it is also present in a select variety of bacteria, most of
which are facultative anaerobes. A striking observation is
that these same PPDK-containing bacteria also encode
the DUF299 gene, the same large domain of sequence that
comprises most of the maize RP polypeptide (see Fig. 3).
Since most of the PPDK-DUF299 bacterial species are facultative anaerobes, it is notable that PPDK provides a far
different function in these bacteria than it does in C4 plants.
For example, it has been proposed that the enzyme supplants pyruvate kinase in the glycolytic pathway for anaerobic synthesis of cellular ATP [24]. It is possible that a
10
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‘‘prokaryotic’’ RP, as the DUF299 gene product, evolved
in bacteria in order to regulate the ATP synthesizing function of PPDK. Since the DUF299 contains a GTP-binding
domain it is reasonable to speculate that RP evolved in
bacteria to down-regulate PPDK activity via an ATPdependent phosphorylation of the active-site Thr (also
strictly conserved in bacterial PPDKs), as opposed to the
ADP-dependent inactivation-phosphorylation mechanism
that RP catalyzes in plants. In such a regulatory scheme,
when cellular ATP begins to accumulate (e.g., at low
energy demand) an ATP-dependent feedback inhibition
of glycolysis via down-regulation of PPDK would ensure
conservation of PEP during glycolytic ATP synthesis. It
may be significant in this regard that maize RP catalyzes
the ADP-dependent inactivation of PPDK from the nonsulfur purple photosynthetic bacterium Rhodospirillum
rubrum [25].
In plants, PPDK inactivation is dependent on the PPDK
being catalytically His-phosphorylated before it can be
20
A
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240
290
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300
A A G A S G D G V K P I
N F G
Y H
K
Fig. 3. Amino acid sequence alignment of maize RP. The maize RP deduced amino acid sequence was aligned, using the ClustalW alignment program of
the MacVector 8.0 software, with orthologs identified in Agrobacterium tumefaciens AAL41032.1 (At), Geobacter metallireducens ABB33596.1 (Gm),
Listeria monocytogenes AAT04664.1 (Lm), Novosphingobium aromaticivorans ABD24566.1 (Na), Streptococcus agalactiae CAD47374.1 (Sa), and
Symbiobacterium thermophilum BAD39570.1 (St). These bacterial species also contain orthologs of plant PPDK. The first 130 amino acids of the maize RP
sequence have been removed. Regions of amino acid identity are indicated with shading and consensus sequence shown under the aligned sequences.
680
J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680
phosphorylated by the ADP-dependent RP-catalyzed reaction; the catalytic phosphate is derived from ATP [22]. It has
also been reported that PPDK may be catalytically phosphorylated by pre-incubation with PEP [22]. Therefore, it
is logical to suggest that if RP functions to regulate PPDK
activity in bacteria (in the ATP-forming direction) and the
substrate for inactivation is the catalytically phosphorylated
form, it is likely that PEP is the immediate phosphate donor
for catalytic phosphorylation. So it is possible that the ADPdependent inactivation of PPDK evolved in bacteria to regulate PPDK catalyzing the formation of ATP and pyruvate
and that this regulatory mechanism has been adopted by
plants that use PPDK in either direction (i.e., pyruvate- or
PEP-forming). The fact that PPDK needs to be catalytically
phosphorylated before it can be inactivated may have
evolved to regulate PPDK in the ATP-forming direction
(glycolytic direction) and the true substrate for catalytic
phosphorylation is PEP. In contrast, in plants in which
PPDK operates in the PEP-forming direction ATP provides
the catalytic phosphate. And finally, the nucleotide
substrate specificity of RP has altered from using ATP in
bacteria to using ADP in plants as the function of PPDK
altered. The role of PPDK and RP in microorganisms is currently under investigation and will be discussed elsewhere.
Acknowledgment
J.N.B. wishes to thank Timothy Sexton for technical
assistance.
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