Analysis of the chime-ras constructed indicated that the region from amino acid residue 303 to the C-terminal end of NADP-ME2 is critical for fumarate activation.. However, the region fla
Trang 1regulation of cytosolic Arabidopsis thaliana NADP-malic enzymes
Mariel C Gerrard Wheeler1, Cintia L Arias1, Vero´nica G Maurino2, Carlos S Andreo1and
Marı´a F Drincovich1
1 Centro de Estudios Fotosinte´ticos y Bioquı´micos, Universidad Nacional de Rosario, Argentina
2 Botanisches Institut, Universita¨t zu Ko¨ln, Cologne, Germany
Introduction
Malic enzymes (MEs) catalyse the reversible oxidative
decarboxylation of l-malate to pyruvate, CO2 and
NAD(P)H in the presence of a divalent cation [1] This
enzyme is widely distributed in nature, as the substrates
and products of the reaction participate in different
met-abolic pathways In plants, photosynthetic and
nonpho-tosynthetic NADP-dependent isoenzymes (NADP-ME;
EC 1.1.1.40) have been found in both plastids and cytosol [2] Moreover, the co-expression of different NADP-ME isoenzymes has been observed in the same cell, and even in the same subcellular compartment [3] The elucidation of the biological role of the different NADP-ME isoenzymes, apart from being involved in
C4photosynthesis or crassulacean acid metabolism, will
Keywords
allosteric; Arabidopsis thaliana; isoenzymes;
NADP-malic enzyme; regulation
Correspondence
M F Drincovich, Centro de Estudios
Fotosinte´ticos y Bioquı´micos (CEFOBI),
Universidad Nacional de Rosario, Suipacha
531, 2000 Rosario, Argentina
Fax: 54 341 4370044
Tel: 54 341 4371955
E-mail: drincovich@cefobi-conicet.gov.ar
(Received 19 June 2009, revised 28 July
2009, accepted 4 August 2009)
doi:10.1111/j.1742-4658.2009.07258.x
The Arabidopsis thaliana genome contains four genes encoding NADP-malic enzymes (NADP-ME1–4) Two isoenzymes, NADP-ME2 and NADP-ME3, which are shown to be located in the cytosol, share a remarkably high degree of identity (90%) However, they display different expression patterns and show distinct kinetic properties, especially with regard to their regulation by effectors, in both the forward (malate oxida-tive decarboxylation) and reverse (pyruvate reducoxida-tive carboxylation) reac-tions In order to identify the domains in the primary structure that could
be responsible for the regulatory differences, four chimeras between these isoenzymes were constructed and analysed All chimeric versions exhibited the same native structures as the parental proteins Analysis of the chime-ras constructed indicated that the region from amino acid residue 303 to the C-terminal end of NADP-ME2 is critical for fumarate activation However, the region flanked by amino acid residues 303 and 500 of NADP-ME3 is involved in the pH-dependent inhibition by high malate concentration Furthermore, the N-terminal region of NADP-ME2 is necessary for the activation by succinate of the reverse reaction Overall, the results show that NADP-ME2 and NADP-ME3 are able to distinguish and interact differently with similar C4 acids as a result of minimal struc-tural differences Therefore, although the active sites of NADP-ME2 and NADP-ME3 are highly conserved, both isoenzymes acquire different allo-steric sites, leading to the creation of proteins with unique regulatory mech-anisms, probably best suited to the specific organ and developmental pattern of expression of each isoenzyme
Abbreviations
GFP, green fluorescent protein; ME, malic enzyme.
Trang 2require further effort, as the gene family of this protein
is more complex than expected [4]
The Arabidopsis thaliana genome contains four
NADP-ME genes [3,4] One gene encodes a plastidic
enzyme (NADP-ME4 [3]), but the other three isoenzymes
do not possess predictable organellar sorting sequences
and thus are thought to be located in the cytosol
(NADP-ME1–3) Previous studies have indicated
dif-ferential expression patterns for each isoenzyme [3]
In this regard, although ME2 and
NADP-ME4 are constitutively expressed in mature organs,
NADP-ME1 is restricted to secondary roots and
NADP-ME3 to trichomes and pollen [3] Although
the four isoenzymes share a high degree of identity
(75–90%), the recombinant enzymes show distinct
structural and kinetic properties [3,5] Specifically,
the isoenzymes behave differently in terms of
regula-tion by metabolic effectors, NADP-ME2 being the
most highly regulated, especially by activation [5]
In particular, NADP-ME2 and NADP-ME3 share
90% identity (Fig 1), are encoded in the same
chromosome and belong to the cytosolic dicot group
in a phylogenetic tree constructed with plant
NADP-ME sequences [3] In the malate oxidative decar-boxylation reaction, although NADP-ME2 is highly activated by aspartate, fumarate and succinate, NADP-ME3 is inhibited by fumarate with no modifi-cation of the enzymatic activity in the presence of aspartate and succinate [5] Furthermore, although suc-cinate and fumarate show strong activation of the NADP-ME2 pyruvate reductive decarboxylation reac-tion (up to 400%), these metabolites act as inhibitors
of the ME3 reverse reaction [5] Two NADP-ME2 amino-terminal deletions previously analysed indicated that some residues from this region are criti-cal for aspartate and CoA activation [5] However, regions involved in the differential regulation by fuma-rate and succinate could not be mapped by this approach Moreover, the mutation of R115 in NADP-ME2 indicated that this amino acid residue is involved
in fumarate activation [5] However, this residue is conserved in NADP-ME3, indicating that other amino
Fig 1 Sequence alignment of A thaliana NADP-ME2 and NADP-ME3 Regions of the primary structure of each isoenzyme that are involved in fumarate activation (in yellow), CoA activation (underlined) and malate inhibition (in green) of the forward reaction are highlighted In addition, the regions involved in succinate activation of the reverse reaction are highlighted in light blue Nonconserved residues between the two sequences are shown in bold ‘*’, iden-tical residues; ‘:’, conserved substitution; ‘.’, semiconserved substitution.
Trang 3acid residues are responsible for the differential
regula-tion by fumarate In this work, NADP-ME2 (Uniprot
Accession Number Q9LYG3) and NADP-ME3
(Uniprot Accession Number Q9XGZ0) are
experimen-tally shown to be located in the cytosol; moreover, the
relationship between the primary structure and
differ-ences in regulation was investigated by the
character-ization of complementary chimeras between the two
isoenzymes The segments swapped in the construction
of the chimeras allowed us to evaluate the eight
non-conserved amino acid residues between NADP-ME2
and NADP-ME3 These amino acid residues are
sepa-rated into two regions: three are located in the first
segment swapped and five in the second (Fig 1) Using
this approach, specific segments of the primary
struc-ture responsible for regulatory differences were
identi-fied, indicating that minimal structural changes are
responsible for the distinct behaviour of these two
highly similar NADP-ME isoenzymes
Results
Subcellular localization of A thaliana NADP-ME2
and NADP-ME3
In order to determine the subcellular localization of
A thaliana NADP-ME2 and NADP-ME3, the
full-length cDNA of each isoenzyme was fused in frame to the green fluorescent protein (GFP) coding sequence, and the localization of the fluorescence was assayed by transient expression in A thaliana cell cultures Figure 2 clearly shows that NADP-ME2 and NADP-ME3 are both homogeneously distributed in the cytosol A con-trol assay with the GFP coding region shows the locali-zation of free GFP in the cytosol and nucleus (Fig 2)
Structural characterization of chimeric NADP-MEs
In order to examine the sequence domains responsible for regulatory differences between the cytosolic isoen-zymes NADP-ME2 and NADP-ME3, four chimeric proteins (named ME2.3, ME2.3¢, ME3.2 and ME3.2¢; Fig 3) were successfully expressed in E coli and puri-fied to homogeneity To determine whether the chime-ric proteins display any structural changes in relation
to the parental proteins, CD spectra for all chimeric and parental enzymes were compared In all cases, the
CD spectra obtained after corrections for protein con-centration were very similar (data not shown), indicat-ing that there was no significant loss of secondary structure in the chimeric proteins
Monomeric molecular masses of 65 kDa were determined by SDS-PAGE for all chimeras (data not
Fig 2 Subcellular localization of A thaliana NADP-ME2 and NADP-ME3 Transient expression of ME2::GFP, 35S::NADP-ME3::GFP and 35S::GFP in A thaliana protoplasts Bright field images with the superimposed GFP fluorescence images shown at the top Fluorescence distribution is shown at the bottom The scale bar represents 12 lm.
Trang 4shown), which are in accordance with those
obtained for the parental recombinant proteins [3]
Native electrophoresis of the purified proteins
indi-cated that the parental and chimeric proteins
presented almost identical electrophoretic mobility (data not shown and [3]) Moreover, the parental and chimeric proteins presented highly similar native molecular masses by gel filtration chromatography,
Fig 3 Chimeric NADP-MEs constructed and analysed in the present work The conserved restriction sites EcoRV and BclI at positions 910 and 1500, respectively, of the cDNA of parental enzymes (NADP-ME2 and NADP-ME3) were used to construct the complementary chimeric enzymes ME2.3, ME2.3¢, ME3.2 and ME3.2¢ These sites correspond to positions 303 and 500, respectively, in the protein sequence of NADP-ME2 and NADP-ME3 The recombinant NADP-MEs that are activated by fumarate or CoA or inhibited by high malate concentration
at pH 7.0 (for the forward reaction) or that are activated by succinate (for the reverse reaction) are indicated on the right by ‘4’ Regions of the primary structure of each parental NADP-ME that are involved in fumarate, CoA and succinate activation and malate inhibition are indicated.
Table 1 Properties of parental and chimeric NADP-MEs The indicated values are the average of at least three different measure-ments ± SD For kcatcalculations, a 65 kDa monomeric molecular mass was used for all isoenzymes Some values for parental NADP-ME2 and NADP-ME3, obtained previously [3], are included for comparison (k catD ⁄ k catC , k cat decarboxylation ⁄ k cat carboxylation ; M, monomeric molecular mass; N, native molecular mass; NI, no inhibition was observed.).
Malate oxidative decarboxylation, pH 7.5
Malate oxidative decarboxylation; pH 7.0
Pyruvate reductive carboxylation; pH 7.0
Relation forward ⁄ reverse reaction
Structural properties
a
Determined by SDS-PAGE.bDetermined by gel filtration chromatography.cAt pH 8.0, as inhibition by high malate concentration was observed at values lower than pH 8.0.
Trang 5which were consistent with tetrameric oligomeric
states (Table 1)
Kinetic characterization of chimeric NADP-MEs in
the oxidative decarboxylation direction
Previous results have indicated that NADP-ME2
dis-plays higher decarboxylation activity at a lower pH
(optimum pH 6.8) than NADP-ME3 (optimum pH
7.7) [5] Nevertheless, as at pH 7.5 both isoenzymes
retained 95% of the maximal activity (data not
shown), this pH was chosen for the comparative
char-acterization of the kinetic and regulatory properties of
the chimeric proteins (Table 1) As ME3.2¢ was
inhib-ited by high malate concentration at pH values lower
than pH 8.0 (data not shown), the kinetic analysis of
this chimera was performed at pH 8.0 (Table 1)
All chimeras showed less specific activity than the
parental isoenzymes (Table 1) The kcatvalues obtained
were between 48% and 14% of the value obtained for
NADP-ME2, the parental enzyme with the highest
specific activity (Table 1) The KmNADP and Kml-malate
values of the chimeric proteins were of the same order
of magnitude as those of NADP-ME2 (Table 1) These
results indicate that, despite the differences detected,
the binding sites for the substrates were integral in the
chimeric proteins
Regulatory properties of the chimeric NADP-MEs
in the oxidative decarboxylation direction
Several compounds were tested as possible effectors of
the enzymatic activity of each chimeric NADP-ME in
the direction of the oxidative decarboxylation of
l-malate (Fig 4), and compared with the results
obtained with the parental isoenzymes [5] With the
exception of acetyl-CoA and CoA, the effectors were
tested at two concentrations, 0.5 and 2 mm, which are
referred to as low and high concentrations, respectively
As in the case of NADP-ME2 and NADP-ME3,
oxaloacetate and ATP were the strongest inhibitors of
the enzymatic activity of all chimeric proteins (Fig 4)
ME2.3¢ and ME3.2 were inhibited only by high ATP
concentration, whereas all other chimeras were
inhib-ited by both high and low ATP concentrations,
proba-bly because of the higher affinity for this inhibitor
Glucose-6-phosphate also inhibited the enzymatic
activity of all proteins (Fig 4), although oxaloacetate
and ATP were the strongest inhibitors
The effects of acetyl-CoA and CoA on the
enzy-matic activity were tested at 20 lm (Fig 4)
Acetyl-CoA did not modify significantly the activity of the
chimeras However, CoA maintained its status as an
activator in the chimeric proteins ME2.3¢ and ME3.2, whereas no modification of ME2.3 and ME3.2¢ activi-ties were observed in the presence of this compound (Fig 4)
Furthermore, both succinate and aspartate were able
to activate all chimeric enzymes, although activation
by succinate was observed only at high concentration
in the case of ME2.3 and ME3.2¢ (Fig 4) By contrast, fumarate activated only ME3.2 and inhibited ME3.2¢, but only at high concentration in the latter case (Fig 4)
Kinetic characterization of chimeric NADP-MEs in the oxidative decarboxylation direction at pH 7.0 The effect of the substrate l-malate on the forward reaction of NADP-ME2 and NADP-ME3 was analy-sed at pH 7.0 The kinetic measurements showed that NADP-ME3 was partially inhibited by high concentra-tions of l-malate at this pH (Fig 5) The kinetic data obtained for NADP-ME3 at pH 7.0 fitted to an equa-tion in which two different sites for malate, one cata-lytic and one allosteric, are considered (see Materials and methods [6]) When occupied, the allosteric site decreases the activity of the enzyme, rendering a par-tial inhibition that is characterized by a Krvalue of 0.6 and u value of 0.1 (Table 1) The inhibition of NADP-ME3 by high malate concentration was pH dependent,
as no inhibition was observed at pH 7.5 (Table 1 and [3]) However, NADP-ME2 was not inhibited by high malate concentration at pH 7.0 (Fig 5)
In order to identify sequence segments in the pri-mary structure of NADP-ME2 and NADP-ME3 responsible for the differential behaviour at pH 7.0, the chimeric proteins were analysed at this pH The results indicated that only ME2.3 and ME3.2¢ were inhibited by high malate concentration The data obtained for these enzymes at pH 7.0 fitted well to the equation that considers that the enzyme binds malate
at two different sites, one catalytic and the other allo-steric, as in the case of NADP-ME3 (data not shown) The Kr values obtained (Table 1) indicate that the malate allosteric site of ME2.3 and ME3.2¢ displays lower affinity than that of the parental enzyme NADP-ME3 In turn, the higher u parameters for the chimeras indicate a smaller decrease in the catalytic activity when the allosteric site is occupied by the inhibitor, in comparison with the parental enzyme NADP-ME3 (Table 1) In the case of ME3.2¢, the inhi-bition by high malate concentration was also observed
at pH 7.5 (not shown), but not at pH 8.0, which was used for the kinetic characterization of the chimera (Table 1)
Trang 6Reversibility of the reaction catalysed by chimeric
NADP-MEs
The four chimeric NADP-MEs were tested for their
capability to catalyse the reverse reaction: the
pyru-vate reductive carboxylation As the carboxylation
reaction catalysed by the parental isoenzymes showed
an optimum at pH 7.0 [5], this pH value was used for kinetic analysis of the chimeras All chimeric pro-teins showed less specific activity than NADP-ME3 (Table 1) ME2.3 and ME3.2¢ are the chimeras with the highest kcat values for the reverse reaction, with
Fig 4 Regulatory properties of the chimeric NADP-ME isoenzymes in the oxidative decarboxylation direction NADP-ME forward activity was measured for each isoenzyme at pH 7.5 in the absence or presence of 0.5 or 2 m M of each effector [indicated as Succinate 0.5 or 2; Fumarate 0.5 or 2; Asp (aspartate) 0.5 or 2; OAA (oxaloacetate) 0.5 or 2; ATP 0.5 or 2; Glucose 6P (glucose-6-phosphate) 0.5 or 2] or 20 l M
of CoA or acetyl-CoA The results are presented as the percentage of activity in the presence of the effectors in relation to the activity mea-sured in the absence of the metabolites for each of the respective enzyme constructs The assays were performed at least in triplicate and the error bars indicate SD Significant inhibition (as indicated in Materials and methods): dark grey and single-hatched bars Significant activa-tion (as indicated in Materials and methods): light grey and double-hatched bars The results for parental NADP-ME2 and NADP-ME3, obtained previously [5], are included for comparison.
Trang 7values even higher than that of NADP-ME2
(Table 1) These proteins were able to catalyse the
reductive carboxylation reaction at higher rates than
the oxidative decarboxylation reaction (Table 1)
However, ME2.3 and ME3.2¢ display the lowest
affin-ity towards pyruvate in comparison with all the other
isoenzymes (Table 1)
Regulatory properties of the chimeric NADP-MEs
in the reductive carboxylation direction
The effect of several metabolites on the reductive
carboxylation reaction of the chimeric proteins was
analysed and compared with the results obtained with
the parental enzymes (Fig 6)
l-Malate, one of the products of the reverse
reac-tion, was the strongest inhibitor of the enzymatic
activ-ity of all the chimeric versions (Fig 6) Aspartate also
inhibited the reductive carboxylation of all chimeras
and NADP-ME2, but did not modify the enzymatic
activity of NADP-ME3 (Fig 6)
With regard to succinate, this organic acid activated
the chimeric enzymes possessing the amino-terminal
region of NADP-ME2, ME2.3 and ME2.3¢ (Fig 6)
By contrast, succinate did not modify the activity of
ME3.2 and ME3.2¢ (Fig 6) In the case of fumarate,
all chimeras showed activation by this compound
(Fig 6), as did NADP-ME2, whereas the parental
iso-enzyme NADP-ME3 was inhibited by both succinate
and fumarate [5]
Discussion
A thaliana NADP-ME2 and NADP-ME3 are shown
to be located in the cytosol The measurement of
enzymatic activity in the presence of several putative
metabolic effectors indicated distinct regulatory pat-terns for both isoenzymes (Figs 4–6) In order to iden-tify the key sequence regions associated with the more relevant kinetic differences between these highly similar isoenzymes, several chimeras of these proteins were constructed and analysed All the chimeric proteins showed structural integrity by CD analysis and conser-vation of the quaternary conformation (Table 1) Thus, the absence of severe structural changes with respect to the parental enzymes, and the fact that the chimeras were functional proteins (Table 1), allowed
us to use them as a tool to compare regulatory pat-terns and to evaluate the determinants of the primary sequence associated with them
Regulatory regions associated with fumarate and CoA activation of the malate oxidative decarboxylation reaction of NADP-ME2 Several compounds were tested as possible modifiers of the malate oxidative decarboxylation reaction cataly-sed by NADP-ME2 and NADP-ME3 Oxaloacetate, ATP, glucose-6-phosphate and acetyl-CoA similarly affected the activity of both native enzymes and chime-ras (Fig 4 [5]) However, succinate, fumarate, aspar-tate and CoA produced differential effects on the activity of NADP-ME2 and NADP-ME3, as well as the different chimeras analysed (Fig 4)
Fumarate activation was observed for the parental NADP-ME2 and the chimeric enzyme ME3.2 (Fig 4) These proteins share a common region, which extends from amino acid residue 303 to the C-terminal end of NADP-ME2, suggesting that this sequence is associ-ated with the activation mechanism by this compound (Figs 1 and 3) Moreover, amino acid residues from both segments swapped (from amino acid residue 303
Fig 5 NADP-ME2 and NADP-ME3 forward activity as a function of malate concentration at pH 7.0 Free Mg2+and NADP concentrations were kept constant at 10 and 1.0 m M , respectively, in all cases A typical result is shown from at least three independent determinations The data were fitted to the Michaelis–Menten equation for NADP-ME2 or to the model described in Materials and methods for NADP-ME3 (see equation in [6]), and are presented as the percentage of maximum activity The absolute values corresponding to 100% of activity are
497 and 218 UÆmg)1 for NADP-ME2 and NADP-ME3, respectively Malate inhibition was not observed for either isoenzyme at pH 7.5 (Table 1 and [3]).
Trang 8to 500 and from amino acid residue 500 to the
car-boxyl-terminal end of NADP-ME2; Figs 1 and 3) are
involved in this regulation, as the chimeras ME2.3¢
and ME 3.2¢, both bearing only one of these segments,
are not activated by fumarate (Fig 3)
Like NADP-ME2, human mitochondrial
NAD(P)-ME is allosterically activated by fumarate [7] In this case, fumarate binds at the dimer interface, where four amino acid residues are involved: R67, R91, E59 and D102 [7–9] Only two of these amino acid
Fig 6 Regulatory properties of the chimeric NADP-ME isoenzymes in the reductive carboxylation direction NADP-ME reverse activity was measured for each isoenzyme at pH 7.0 in the absence or presence of 1, 7.5 or 15 m M of each effector [indicated as L -malate, Succinate, Fumarate and Asp (aspartate) 1, 7.5 and 15 m M ] The results are presented as the percentage of activity in the presence of the effectors in relation to the activity measured in the absence of the metabolites, for each of the respective enzyme constructs The assays were performed at least in triplicate, and error bars indicate SD Significant inhibition (as indicated in Materials and methods): dark grey and single-hatched bars Significant activation (as indicated in Materials and methods): light grey and double-single-hatched bars The results for parental NADP-ME2 and NADP-ME3, obtained previously [5], are included for comparison.
Trang 9residues (R91 and D102; homologous to R115 and
D126 of NADP-ME2, respectively) are conserved in
A thaliana NADP-ME2 (Fig 1 and [5]), suggesting
that the mechanism of activation should be different
between the two isoenzymes Moreover, these two
amino acid residues are also conserved in
NADP-ME3 (Fig 1), which is not activated by fumarate
(Fig 4) Therefore, other amino acid residues
differ-ent from those proposed for the human isoenzyme
[7–9] are necessary to control the binding capacity
and fumarate response of NADP-ME2 Several
amino acid residues in this C-terminal region (Figs 1
and 3) are good candidates to be involved in this
activation, and their role remains to be determined
by mutational studies Specifically, from the five
non-conserved amino acid residues in the suggested
domain involved in fumarate activation, the
muta-tions at posimuta-tions 357 and⁄ or 360 could be involved
in the differential regulation, as well as the conserved
change at position 543 (Fig 1)
The activation of NADP-ME2 by fumarate could be
relevant in vivo, as A thaliana accumulates large
amounts of fumarate and malate during the day and
uses these organic acids as a way to transport carbon
to other organs, and as energy and carbon sources in
conditions of energy demand [10,11] In this sense, the
activation by fumarate of NADP-ME2, which is
expressed in photosynthetic and nonphotosynthetic
organs of A thaliana, may be linked to the higher
utilization of organic acids on energy demand by the
activation of this isoenzyme when the fumarate
con-centration increases However, our data suggest that
NADP-ME3, which is restricted to pollen and
tri-chomes, is not linked to this organic acid utilization
and regulation
By contrast, the region between amino acid residues
303 and 500 of NADP-ME2 may be associated with
CoA activation of the l-malate decarboxylation
reac-tion because only NADP-ME2, ME2.3¢and ME3.2
showed activation by this compound (Figs 1 and 3)
Previous studies have indicated that the deletion of 44
amino acid residues from the amino-terminal region of
NADP-ME2 provides an enzyme that is not activated
at all by CoA [5] Thus, the activation by this
metabo-lite may require the participation of the region flanked
by amino acid residues 303 and 500 of NADP-ME2
interacting with residues from the amino-terminal
region
Although NADP-ME3 is not activated by
aspar-tate and succinate (Fig 4), it is surprising that the
activity of the four chimeras increases in the
pres-ence of both effectors, although to a different extent
(Fig 4) In this way, it can be inferred that the
acti-vation by these metabolites is mediated by several amino acid residues, not found in NADP-ME3, but distributed in the different protein segments of NADP-ME2 that were swapped by the construction
of the chimeras
Regulatory region associated with the pH-dependent malate inhibition of NADP-ME3 Malate inhibition of the forward reaction at pH 7.0 was observed only in the case of NADP-ME3, ME2.3 and ME3.2¢ (Fig 3, Table 1) These proteins share a common region, which extends from amino acid resi-dues 303 to 500 of NADP-ME3, suggesting that this sequence is associated with the mechanism of substrate inhibition (Figs 1 and 3) The fact that the inhibition
by high substrate concentration was associated with a limited region of the protein supports the hypothesis
of the existence of an allosteric site responsible for such regulation, as in the case of maize photosynthetic NADP-ME [6] In agreement with this, the kinetic data obtained for the enzymes that were inhibited by l-malate fitted very well to the equation that considers
an allosteric inhibitor binding site for malate (Fig 4 [6]) Moreover, as the inhibition by malate is pH dependent (Table 1), it is concluded that the amino acid residue(s) involved in this allosteric regulation may change the protonation state between pH 7.0 and
pH 7.5, leading to the loss of inhibition at higher pH
In the particular case of ME3.2¢, the loss of malate inhibition is observed at higher pH (pH 8.0, Table 1)
It is thus possible that in this chimera a change in the pKa value of the amino acid residue(s) involved in malate inhibition may occur as a result of interaction with different amino acid residues in the allosteric site The amino acid residue changes at positions 357, 420 and⁄ or 481 between NADP-ME2 and NADP-ME3 are good candidates for involvement in the pH-dependent regulation by malate, as they involve changes from noncharged amino acid residues in NADP-ME2 to positive amino acid residues, depending on pH, in NADP-ME3 (Fig 1)
The inhibition by excess l-malate was marked as a pH-dependent characteristic of MEs implicated in C4 photosynthesis [6] This in vivo regulatory mechanism was suggested to occur through the pH change induced
in illuminated chloroplasts, and ensures that
NADP-ME is fully active only at pH 8.0, when carbon fixa-tion is in progress Thus, the pH-dependent malate inhibition of NADP-ME3 was unexpected, as it is a cytosolic isoenzyme not implicated in photosynthesis
In this way, this isoenzyme from a C3 species displays the feature of malate inhibition at pH 7.0 associated
Trang 10with C4 photosynthesis, probably as an evolutionary
ancestor of C4 NADP-ME Similarly, a
nonphoto-synthetic recombinant NADP-ME from tobacco also
showed partial inhibition by l-malate [12] Further
studies should be conducted to reveal whether the
pH-dependent regulation of a nonphotosynthetic
isoenzyme may be relevant in vivo, especially with
regard to the localization of NADP-ME3 in the
cyto-sol of pollen and trichome cells
Regulatory region associated with succinate
activation of the pyruvate reductive
carboxylation reaction of NADP-ME2
The activation of the pyruvate reductive carboxylation
reaction by succinate was only observed in the case of
NADP-ME2 and the chimeras ME2.3 and ME2.3¢
(Fig 6) Thus, a regulatory region associated with
acti-vation by this metabolite could be defined, which
com-prises the first 303 amino-terminal amino acid residues
of NADP-ME2 (Figs 1 and 3) However, the three
nonconserved amino acid changes between
NADP-ME2 and NADP-ME3 are located in the
amino-termi-nal region (Fig 1), which is not involved in succinate
activation, as NADP-ME2 lacking the first 44 amino
acid residues is still activated by succinate [5] Thus,
some of the semiconserved or conserved amino acid
residue changes may be involved in succinate
activa-tion Good candidates are the changes in charge at
positions 253 and⁄ or 295 (Fig 1)
By contrast, fumarate was able to activate the
reverse reaction catalysed by NADP-ME2 (Fig 6 [5])
However, although NADP-ME3 is not activated by
this compound at all, it is surprising that the activity
of the four chimeras is increased by fumarate (Fig 6)
In this regard, ME3.2¢, the chimera that shares the
minimum number of amino acid residues with
NADP-ME2, is less activated by this compound than the
other compounds (Fig 6) In this way, amino acid
res-idues of different segments of NADP-ME2 swapped in
the construction of the chimeras are involved in the
fumarate activation of the pyruvate reductive
carboxyl-ation reaction However, the different degree of
fuma-rate activation shown by the chimeras (e.g 150% in
the case of ME3.2 and 671% in the case of ME2.3¢
with 7.5 mm of fumarate) may indicate that some
regions are more critical than others in the activation
by this allosteric modulator This hypothesis should be
tested by site-directed mutagenesis of candidate amino
acid residues from the different regions and by
estima-tion of the kinetic parameters of fumarate activaestima-tion
Aspartate inhibits NADP-ME2 and all the chimeras
analysed, but is unable to decrease NADP-ME3
activ-ity, although tested at high concentration (Fig 6) These results are consistent with an allosteric type of inhibition, in which amino acid residues from the dif-ferent segments used to construct the chimeras are involved However, as some chimeras are inhibited only at high aspartate concentrations (e.g ME3.2¢ is not inhibited at all at 1 mm aspartate), some segments
of the primary structure seem to be more critical than others (Fig 6) This hypothesis can be tested by site-directed mutagenesis of candidate amino acid residues from the different regions, and by estimation of the affinity towards aspartate as inhibitor
The forward and reverse reactions are distinctly regulated by effectors which are associated with different protein determinants
Finally, the results obtained suggest that the regulation
of the forward and reverse NADP-ME activities is mediated by different protein regions (Figs 4 and 6)
In this sense, the dual effect of aspartate in the NADP-ME2 reaction, which activates the decarboxyl-ation and inhibits the carboxyldecarboxyl-ation reaction, can be explained through a conformational change in the enzyme induced by the substrate malate, which can be important for exposing an aspartate-activating binding site [5]
By contrast, succinate and fumarate strongly increased the activity of NADP-ME2 in both direc-tions of the reaction (Figs 4 and 6) However, despite the structural similarity between these two organic acids, the kinetic results indicated that the activation
by these compounds was mediated by different bind-ing sites This conclusion is supported by previous studies, which showed that R115 mutation of NADP-ME2 abolished the activating effect of fumarate, but did not modify the activity measured in the presence
of succinate [5] In turn, our experimental data clearly indicate that, for each separate metabolite, the regula-tion of the direct reacregula-tion is mediated by different sites than in the reverse reaction (Fig 3) Again, conformational changes induced in the protein by l-malate or pyruvate could provide an explanation for these observations A future challenge will be to determine the three-dimensional structure of a plant NADP-ME in the presence and absence of the sub-strates to analyse the conformational changes that are induced by the binding of the substrate, which may influence the observed allosteric regulation of each isoenzyme As suggested recently, a knowledge of the allosteric interactions could be very useful in protein design inhibition or activation to influence protein function as required [13]