From the Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), CONICET, Fund. M. Lillo, Universidad Nacional de Rosario, Suipacha 531, Rosario 2000, Argentina
Received for publication, December 9, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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Malic enzymes catalyze the oxidative
decarboxylation of L-malate to yield pyruvate,
CO2, and NAD(P)H in the presence of a bivalent metal ion.
In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are
involved in a wide range of metabolic pathways. The
C4-specific NADP-ME has evolved from C3-type
malic enzymes to represent a unique and specialized form of NADP-ME as
indicated by its particular kinetic and regulatory properties. In the
present study, the mature C4-specific NADP-ME of maize was
expressed in Escherichia coli. The recombinant enzyme has
essentially the same physicochemical properties and
Km for the substrates as those of the naturally
occurring NADP-ME previously characterized. However, the
kcat was almost 7-fold higher, which may
suggest that the previously purified enzyme from maize leaves was
partially inactive. The recombinant NADP-ME also has a very low
intrinsic NAD-dependent activity. Five mutants of NADP-ME
at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V,
A392G, A387G, and R237L) were constructed by site-directed mutagenesis
and purified to homogeneity. The participation of these residues in
substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular dichroism and intrinsic fluorescence spectra.
The results obtained were compared with a predicted three-dimensional model of maize C4 NADP-ME based on crystallographic studies
of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expression of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the
binding of substrates and/or catalysis.
Malic enzymes
(ME)1 catalyze the oxidative
decarboxylation of L-malate to yield pyruvate,
CO2, and NAD(P)H in the presence of a bivalent metal ion.
They are widely distributed in nature, being found in bacteria, fungi,
animals, and plants. In eukaryotic organisms, different ME isoforms are
found in the cytosol, mitochondria, or chloroplasts. As the products of
the ME reaction can support diverse biological processes, the enzyme is
involved in a great number of metabolic pathways depending on the
tissue and subcellular localization. Most ME can use only NAD or NADP
as essential cofactors. Thus, they are classified as NAD (EC 1.1.1.38
or EC 1.1.1.39, depending on the ability to decarboxylate
oxaloacetate)- or NADP (EC 1.1.1.40)-dependent malic enzymes.
In plants, different isoforms of the NADP-dependent malic
enzyme (NADP-ME) are involved in a wide range of metabolic pathways (1). The photosynthetic C4 NADP-ME, which is involved in
the CO2 concentrating mechanism that increases the
photosynthetic yield of NADP-ME C4 plants, is
compartmentalized in bundle sheath chloroplasts. In NADP-ME
C4 plants, these organelles show a gradation of structure
from chloroplasts with rudimentary grana (in maize and crabgrass) to
completely agranal (in sugarcane and sorghum) (2). It was recently
suggested that the high level activity of the C4 NADP-ME
could strongly affect the development of the bundle sheath
chloroplasts, as transgenic rice expressing the C4 maize
NADP-ME also showed an aberrant structure (3).
The C4-specific NADP-ME represents a unique and specialized
form of ME as indicated by its particular kinetic and regulatory properties (1, 4). As other C4-specific enzymes, NADP-ME pre-existing genes of the C3-type were probably recruited
for operating in the C4 pathway after acquiring diverse
regulatory elements and unique kinetic and regulatory properties (1,
5-7). Nevertheless, all plant NADP-MEs are highly homologous and share several conservative sites (from I to V; see Ref. 1). Interestingly, two of these sites (site II and V) present the typical signature motif
GXGXXG, which is diagnostic, along with
other characteristics, also shared by these sites, of a
dinucleotide-binding fold (8, 9).
Recently, crystal structures of human mitochondrial
NAD(P)-dependent malic enzyme (EC 1.1.1.39; see Ref. 10),
pigeon cytosolic NADP-malic enzyme (EC 1.1.1.40; see Ref. 11), and
Ascaris suum mitochondrial NAD-ME (EC 1.1.1.38; see Ref. 12)
were resolved. The structures revealed that malic enzymes belong to a
new class of oxidative decarboxylases (10). Although the amino acid
residue sequences of the human mitochondrial NAD(P)-ME, the pigeon
cytosolic NADP-ME, and the maize chloroplastic C4 NADP-ME are highly conserved (between 44 and 50% identity), there are several
kinetic differences among them. For example, human NADP-ME shows dual
specificity for NAD and NADP, cooperativity in the binding of malate
and regulation of activity by fumarate and ATP; pigeon NADP-ME tetramer
presents half-of-the-sites reactivity; and maize NADP-ME is inhibited
by malate only at acidic pH.
In the present work, the C4-specific NADP-ME from maize was
expressed in Escherichia coli as a functional protein. The
recombinant mature enzyme shows almost all the expected physicochemical
and kinetic properties of the natural enzyme, and was used to perform site-directed mutagenesis of residues near or at conserved site II or V
of plant NADP-MEs (1). The kinetic and structural properties of the
mutated proteins in relation of a predicted three-dimensional model
based on the crystal structure obtained for the human and pigeon
NAD(P)-ME were analyzed. The data presented here represent the first
prokaryotic expression of a plant NADP-ME and reveals valuable insight
into the structure of the active site.
Cloning of Maize C4 NADP-ME and Construction of a
Vector for Expressing the Mature Protein--
A random primed partial
cDNA of maize C4 NADP-ME (1.3 kb from the 3'-terminal
cDNA provided by T. Nelson, Yale University) was used as
hybridization probe to screen a
To make a plasmid construction for expressing the mature C4
NADP-ME protein, oligonucleotides primers (5EM,
5'-CATGCCATGGCGATGGTCTCCAACG-3' and 3EM,
5'-ACGCTCGAGGCACTACCGGTAGTTGCG-3') containing the first codon of the
mature maize C4 NADP-ME after transit peptide cleavage (predicted by ChloroP V1.1 Prediction Program and according to Ref. 14)
and the stop codon, respectively, were used to perform a PCR using
pBS-ME as template. The primers also introduced unique NcoI
and XhoI sites at the 5' and 3' of the NADP-ME insert to easily subclone the PCR fragment into the pET32 expression vector (Novagen). The construction obtained was designed in such a way that,
after enterokinase digestion of the recombinant quimeric protein, only
three extra amino acids are introduced at the NH2 terminus
of the mature C4 NADP-ME. The insert in the expressed vector obtained (pET32-ME) was sequenced to be sure that no errors were
introduced because of PCR and subcloning procedures.
Complementation of an E. coli Mutant by pBS-ME--
The pBS-ME
plasmid was studied regarding the complementation of an E. coli mutant lacking NAD(P)-ME and phosphoenolpyruvate carboxykinase activity (JM1321 strain, Ref. 16). In this way, EJ1321,
EJ1321(pBS), and JM1321(pBS-ME) (not transformed and transformed with
pBS or pBS-ME, respectively) were grown on minimum media (SPI: 15 mM (NH4)2SO4, 80 mM K2HPO4, 44 mM
KH2PO4, and 3 mM sodium citrate)
supplemented or not with 0.4% L-malate or 0.4% glucose in
the presence of 25 µg/ml streptomycin to study the ability of the
bacteria to grow with C4 acids as the sole carbon source. The presence of colonies and the absorbance at 600 nm was measured in
liquid media after 24-36 h of incubation at 37 °C as criteria of growth.
Expression and Purification of Recombinant C4
NADP-ME--
In the pET32-ME expression vector, the mature
C4 NADP-ME was connected in-frame with the His tag to
facilitate purification of the expressed fusion protein by a
nickel-containing His-Bind column (Novagen). The BL21(DE3) E. coli strain transformed with pET32-ME was grown on LB medium in
the presence of 100 µg/ml ampicillin until the culture reached an
A600 of 0.6. The inductor lactose (2% w/v) was
added and the bacteria were cultured for a further 16 h at
30 °C. The cells were then harvested by centrifugation for 5 min at
4,000 × g; resuspended in buffer A (0.5 M
NaCl, 20 mM Tris-HCl, pH 7.9) containing 5 mM
imidazole, 0,01 µg/µl leupeptin, and 2 mM
phenylmethylsulfonyl fluoride; sonicated and centrifuged for 10 min at 7,000 × g at 4 °C. The supernatant (soluble
fraction) was used for protein purification onto a nickel-NTA column
according to the protocol provided by the manufacturer (Novagen), but
using 200 mM imidazole for elution. The enzyme was then
concentrated on Centricon YM-30 (Amicon) and desalted according to
Penefsky (17) using buffer B (100 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0.1 mM EDTA, 20% (v/v)
glycerol, and 20 mM
Purified fusion NADP-ME protein was then incubated with enterokinase
(1:100) in buffer B at 10 °C for 2 h to remove the
NH2 terminus codified by the expression vector. The protein
was further purified using an affinity Affi-Gel blue column (Bio-Rad)
followed by a second nickel-NTA column. The purified enzyme was stored at Site-directed Mutagenesis of C4
NADP-ME--
Site-directed mutagenesis of maize mature NADP-ME was
carried out according to the procedures described by Mikaelian and
Sergeant (19). The oligonucleotides primers that introduced mutations in the wild-type protein were as follows: Gsite2V,
5'-TTGGTACTTGTAGATTTGGTTTGTCAG-3'; Gsite5V,
5'-AGTTACAGCCTCCACAGCAACAAGGAA-3';
A392G, 5'-AGAGCAATAAGTTCTCCAATACCAGT-3'; A387G,
5'-TCCACCCTCCCCAGCACC-3'; R237L,
5'-ACTGATGGTGAGCTAATCTTGGGA-3'; in which
the mutation positions are highlighted and underlined in the
oligonucleotide sequence.
The PCR was carried out using a mixture of Taq and
Pfu polymerases (10:1) to reduce possible mistakes
introduced by Taq polymerase. The fragments containing the
mutations were subcloned from the PCR products to the pET32-ME,
replacing the wild-type sequence. The introduced sequences in the
mutated plasmids were sequenced to verify the introduction of the
corresponding mutations and that no errors were added because of PCR
and subcloning procedures. The mutant proteins were expressed and
purified as previously indicated for the wild-type NADP-ME, except that
the Affi-Gel blue column was omitted in some cases because of the lack
or low binding of the mutated enzymes.
NADP-ME Activity Assays--
NADP-ME activity was determined as
described by Maurino et al. (15). Initial velocity studies
were performed by varying the concentration of one of the substrates
around its Km while keeping the other substrate
concentrations at saturating levels. All kinetic parameters were
calculated at least by triplicate determinations and adjusted to
nonlineal regression. Free concentrations of all substrates were
calculated according to Grover et al. (20). The
optimal pH of the recombinant NADP-ME reaction was determined using
different buffers for various pH ranges: 50 mM Mes (pH
5.5-6.5), 50 mM Tricine-Mops (pH 7.0-7.5), and 50 mM Tris-HCl (pH 7.5-8.5).
Malic enzyme activity in the presence of NAD was measured in a
reaction mixture to keep free Mg2+
concentration constant as the NAD level increased (as described in Ref.
21). In this case, the reaction medium contained 50 mM
Tris-HCl (pH 7.0), 10 mM Mg Cl2, and 4 mM L-malate. Initial activity was measured at
different NAD concentrations by adding a mixture of
NAD:MgCl2 (1:0.34) solution adjusted to pH 7.0 to the assay
mixture. This care was not taken for NADP because low concentrations of
NADP were required to saturate the NADP-ME activity.
Gel Electrophoresis--
SDS-PAGE was performed in 10% (w/v)
polyacrylamide gels according to Laemmli (22). Proteins were visualized
with Coomassie Blue or electroblotted onto a nitrocellulose membrane
for immunoblotting according to Burnette (23). Affinity-purified
anti-maize NADP-ME antibodies were used for detection (15). Bound
antibodies were located by linking to alkaline
phosphatase-conjugated goat anti-rabbit IgG according to the
manufacturer's instructions (Sigma).
Native IEF was performed in 5% (w/w) polyacrylamide gels. NADP-ME was
localized by activity staining as described by Maurino et
al. (15). Isoelectric points (pI) of the reactive bands were calculated using the Pharmacia broad pI calibration kit. Protein crude
extracts from maize were prepared as described by Maurino et
al. (15).
Native Molecular Mass Estimation--
The molecular mass of the
recombinant native NADP-ME protein was evaluated by gel-filtration
chromatography on a high performance liquid chromatography system
(Waters Associates) with a BioSep-SEC-S300 column (335 × 7.8 mm,
Phenomenex). The column was equilibrated with 25 mM
Tris-HCl at pH 7.0 or 8.0 and 10% (v/v) glycerol and calibrated using
molecular mass standards. The sample and the standards were applied
separately in a final volume of 50 µl at a constant flow rate of 1 ml/min.
Circular Dichroism (CD) and Intrinsic Fluorescent
Spectra--
CD spectra were made with a Jasco J-810
spectropolarimeter using a 1.0-cm path length cell and averaging five
repetitive scans between 250 and 200 nm. Typically, 50 µg of the
wild-type or mutated NADP-ME in buffer Phosphate (20 mM
NaPi, pH 8.0, 5 mM MgCl2) were used
for each assay. Mean residue ellipticity (
Intrinsic fluorescence (IF) spectra of the recombinant wild-type and
mutated NADP-MEs were analyzed with a PerkinElmer 650-40 fluorescence
spectrophotometer thermostated at 20 °C, using an excitation
wavelength of 295 nm and slit widths of 5 nm. Typically, 5 µg of the
wild-type or mutated NADP-ME in buffer Phosphate were used for each
assay. All spectra were corrected for the buffer absorption and the
Raman spectra of water.
Materials--
NADP, L-malate, Tris, enterokinase,
bovine serum albumin, and reagents for Western blot assays were
purchased from Sigma. DNA modifying enzymes were purchased from Promega
(Madison, WI). All other reagents were of analytical grade.
Cloning, Expression, and Complementation of an E. coli Mutant by a
Fusion Protein of Maize C4 NADP-ME--
The plasmid pBS-ME
obtained after screening a cDNA library from maize leaves with a
partial cDNA of the C4 NADP-ME was studied to determine
whether it was able to express NADP-ME fusion protein in E. coli DH5
To analyze if the fusion NADP-ME immunoreactive protein was expressed
as an active enzyme, NADP-ME activity was measured in soluble protein
extracts from overnight DH5
EJ1321 is a triple mutant of E. coli that lacks NAD and
NADP-malic enzyme and phosphoenolpyruvate carboxykinase activities (16). In this way, it is unable to grow in minimal medium with malate
as the sole carbon source, because it cannot convert C4 acids into C3 compounds. To check if transforming this
strain with pBS-ME restored growth on malate, EJ1321, EJ1321(pBS), and EJ1321(pBS-ME) were grown on minimal medium supplemented with malate or
glucose as the sole carbon source. In the presence of glucose, both
EJ1321 and EJ1321(pBS-ME) were able to grow, as liquid overnight
cultures of these bacteria typically presented A600 of 0.5-0.6. Nevertheless, in the presence
of malate, EJ1321(pBS-ME) was able to grow but EJ1321 and EJ1321(pBS)
were not, as overnight cultures of these bacteria in the presence of
malate presented typically A600 of 0.2-0.3 for
EJ1321(pBS-ME) and 0.01-0.02 for the others. Growth on solid media
supplemented with glucose or malate indicated the same result: the
presence of the plasmid pBS-ME restored the growth in malate for the
E. coli mutant EJ1321.
Expression, Purification, and Characterization of Recombinant
Mature Maize C4 NADP-ME--
After removing the region
encoding for chloroplastic transit peptide of the NADP-ME in pBS-ME,
the sequence codifying the mature NADP-ME was cloned in-frame in the
pET-32 expression vector. The E. coli BL21 strain
transformed with pET32-ME was able to overexpress a protein of 79 kDa
after induction with lactose (Fig. 1B). The protein was
found in both soluble and insoluble fractions of BL21(pET32-ME) protein
extracts and reacted with anti-maize NADP-ME-purified antibodies (Fig.
1C). The calculated molecular mass of the expressed protein
corresponded to the expected molecular mass of the fusion protein as
follows: mature maize NADP-ME (62 kDa) plus 17 kDa was codified by the
expression vector.
The 79-kDa fusion protein obtained was purified by an His-Bind affinity
column (Fig. 2A). After
enterokinase digestion of this protein, a 62-kDa protein was obtained
(Fig. 2A). This molecular mass corresponds to the molecular
mass of the mature NADP-ME from maize leaves (14, 15, 25). Moreover,
Western blot analysis of this protein in comparison with protein crude
extracts from maize leaves, indicates the presence of immunoreactive
bands with identical molecular mass (Fig. 2B). As indicated,
the pET32-ME vector was designed in such a way that after enterokinase
digestion of the fusion recombinant protein, the enzyme obtained starts with three amino acid residues codified by the expression vector (Ala-Met-Ala) followed by the amino acid residues of the mature C4 NADP-ME from maize leaves (Met-Val-Ser-). The yield of
the purified recombinant NADP-ME was ~1 mg of soluble protein per 100 ml of liquid culture media.
To verify if the purified recombinant NADP-ME obtained was identical to
the enzyme present in maize leaves, different properties of the enzyme
were studied. Native IEF revealed for NADP-ME activity indicated
the presence of a band with an isoelectric point of 6.7 for the
recombinant NADP-ME (Fig. 2C). Protein crude extracts from
maize leaves presented a band with the same native isoelectric point
(Fig. 2C). The recombinant NADP-ME before enterokinase
digestion presented a more basic active band, probably because of the
amino-terminal fusion fragment (Fig. 2C).
The recombinant NADP-ME obtained was also able to assemble into a
tetrameric form at pH 8.0, as a molecular mass of 226 kDa was
calculated by gel filtration chromatography (not shown). At pH 7.0, the
enzyme eluted in two peaks, one of 216 kDa and a minor peak of 110 kDa
(not shown), which may correspond to the dimeric form of the
C4 maize NADP-ME as previously demonstrated for the purified enzyme from maize leaves at this pH (26).
Analysis of the secondary structure of the recombinant NADP-ME by CD
spectrum showed a peak at 210 nm (Fig.
3A). The IF emission spectrum of the recombinant NADP-ME excited at 295 nm showed a maximum
at 336 nm (Fig. 3B). The purified NADP-ME from maize leaves presented an essentially identical IF spectrum (27).
The apparent kinetic parameters of the recombinant NADP-ME were
measured and compared with the previously published data obtained for
the purified enzyme from maize leaves (Table
I). Although the Km
values obtained for NADP and malate (as well as for Mg2+,
not shown) were in the same order of magnitude as those previously obtained, the kcat of the recombinant enzyme was
more than six times higher than the value previously reported for the
NADP-ME purified from maize leaves (Table I). The recombinant enzyme also presented an optimum pH for activity of 8.0 and was inhibited by
high concentrations of malate at pH 7.0, with Km and Ki values of 0.084 and 4.7 mM,
respectively. Similar values were previously measured for the purified
enzyme from maize leaves (15, 28).
The recombinant NADP-ME also presented activity with NAD; although the
Km value obtained was 103 times higher
and the kcat 15 times lower than the values
obtained in the presence of NADP (Table I). In this case, the enzyme
presented more activity at pH 7.0 than at 8.0.
Characterization of NADP-ME Mutants Near or at the NAD(P)-binding
Sites--
Using the structure of the human mitochondrial NAD-ME and
the pigeon cytosolic NADP-ME as templates (1QR6 and 1GQ2, respectively,
Protein Data Bank), a three-dimensional model of the maize
C4 NADP-ME was predicted by the protein structure
homology-modeling server (SWISS-MODEL). Part of the predicted model,
showing conserved sites II and V of plant NADP-MEs, both of which
present the typical dinucleotide-binding signature motif
GXGXXG, is shown in Fig. 4A. Sites with essentially the
same motif are also found in the human NAD-ME and the pigeon NADP-ME.
The predicted secondary structure shows that site V (which includes
residues 383GAGEAG388 of the maize NADP-ME)
adopts a Rossman
Gly residues at sites II or V of maize NADP-ME were replaced by Val
residues (mutants Gsite2V or Gsite5V for the replacements in sites II
or V, respectively), in an attempt to analyze the involvement of these
two sites in binding NADP. Both mutants were expressed in E. coli and purified to homogeneity, but neither of them displayed
malic enzyme activity although measured at high enzyme and substrate
concentrations. CD spectra of Gsite2V and Gsite5V NADP-ME mutants
indicated that these proteins did not present great variations in
secondary structure in comparison to the wild-type enzyme (Fig.
3A). Essentially the same IF spectra of the wild-type
NADP-ME were also obtained for both Gsite2V and Gsite5V mutants (Fig.
3B).
Three more NADP-ME mutants near or at sites II or V were also
successfully constructed, expressed, and purified to study their kinetic and structural properties. As site V adopts the Rossman
The mutation introduced in A392G has no significant effect on
kcat, but the Km values for
NADP and malate were increased by factors of 3.5 and 2.6, respectively
(Table I). NAD-dependent activity of A392G was also
measured. In this case, the Km and
kcat values were modified by factors of 0.75 and
3.0, respectively, in comparison to the values obtained for the
wild-type NADP-ME (Table I). With regards to the A387G mutant, a
48-fold decrease in the kcat value was measured,
presenting increases of 4.3 and 5.8 times in the Km
values of NADP and malate, respectively, in comparison to the wild-type
enzyme (Table I). NAD-dependent malic enzyme activity could
not be measured in this case, probably because of the low specific
activity of this enzyme (Table I). CD and IF spectra of A392G and A387G
did not present changes from the spectra obtained for the wild-type
NADP-ME (Fig. 3).
The point mutation introduced at site II (R237L) provoked dramatic
kinetic changes in the NADP-ME activity with a 530-fold decrease in
kcat and 36.3- and 15.3-fold increases in
Km values for NADP and malate, respectively.
NAD-dependent malic enzyme was not detected as the case of
R237L (Table I). Wild-type and R237L NADP-MEs presented almost
identical CD and IF spectra (Fig. 3).
Photosynthetic C4 NADP-ME from maize was fully
isolated from a cDNA library and expressed as an active enzyme in
E. coli. The fusion of NADP-ME, along with its chloroplastic
transit peptide, with the To easily purify great amounts of the mature C4-specific
NADP-ME of maize, we constructed, after removing the chloroplastic transit peptide, a vector that allowed us to obtain a recombinant enzyme without any tag or relevant fusion at the amino-terminal region
(after treatment with enterokinase). The NADP-ME obtained showed the
same molecular mass and native isoelectric point of the enzyme present
in maize leaf crude extracts (Fig. 2) and was also able to assemble
into a tetrameric and/or dimeric form depending on the pH, as
previously indicated for the purified enzyme from maize leaves (26).
The kinetic parameters of the recombinant NADP-ME obtained showed that
the Km values for the substrates were quite close to
the values of the native enzyme (Table I). Nevertheless, the enzyme
presented more than six times higher kcat than
previously reported (Table I). This result may be because of partial
inactivation of the enzyme during the purification procedure from maize
green leaves, which takes five steps and several days (28). The fact that the recombinant enzyme stored at 4 °C in buffer B (conditions of the purification procedure) losses nearly 80% of activity in only 2 days indicates that the maximum activity measured for the recombinant
enzyme is more close to the real value for the naturally occurring
enzyme. In this way, the recombinant enzyme obtained in the present
paper is a preferred source for further structural studies and a
rational source for mutagenesis experiments.
Recombinant maize NADP-ME posses also intrinsic NAD-ME activity,
although the kcat/Km value
was 1.5 × 104 times lower in the presence of NAD than
in the presence of NADP (Table I). This result clearly indicates that
the NADP- and NAD-dependent activities previously measured
for the purified enzyme from maize corresponds to the NADP-ME and not
to a co-purification of a NAD-ME, as previously suggested (31). Both
activities are probably catalyzed by the same active site (see below),
as postulated, by kinetic measurements, for the C4 NADP-ME
purified from Flaveria bidentis (21).
The fingerprint region of NAD(P)-binding sites are characterized by a
Gly-rich sequence (GXGXXG), which is the
phosphate binding consensus, an hydrophobic core consisting of six
positions typically occupied by small hydrophobic amino acids, and a
negatively charged residue at the end of the The fact that Gsite2V and Gsite5V resulted in abortive mutants of
NADP-ME is consistent with the three-dimensional model obtained for the
maize C4 NADP-ME that shows that although only the Gly residues at site V adopts a typical Rossman fold, the residues at site
II are also near the nicotinamide ring of the NAD(P) molecule associated (Fig. 4A). In this way, both sites are part of
the NADP-binding site (and the active site) of NADP-ME and that is the
reason for the high degree of conservation among all NAD(P)-ME, discarding the original proposal of two different NAD(P)-binding sites
(one for NADP and the other for NAD) in maize C4 NADP-ME (14).
The three-dimensional structure of maize NADP-ME indicates that site V
(which includes residues 383GAGEAGTGIA392)
adopts a canonical Rossman fold (Fig. 4A, see Ref. 9). Two Ala residues spaced by three amino acids
(GXGXXAXXXA), which may lie on the same side of the helix, were previously suggested as more
characteristics for NADP-binding domains relative to NAD, whereas Gly
residues were preferably found in NAD-dependent enzymes (29, 30, 32). Maize C4 NADP-ME appears to violate this rule in the case of the first Ala residue, as a Gly is found at position 388. Nevertheless, the second Ala residue is found (at position 392),
along with another Ala residue at position 387, which is moved by one
position in relation to the proposed preferably conserved region for
NADP-binding domains. In this way, site-directed mutagenesis of Ala
residues at positions 392 or 387 were performed to test if these amino
acid side chains confer specificity for NADP in C4 maize
NADP-ME. CD and IF spectra of these mutants indicated no major
structural changes in both enzymes (Fig. 3).
Substitution of Ala392 by Gly caused little but consistent
modifications in the values of Km for NADP (and
malate), rendering an enzyme with a decrease of 3.8-fold in the
kcat,NADP/Km,NADP value in relation to the wild-type NADP-ME, decrease
attributed to lower affinity of NADP binding (Table I). Interestingly,
the mutant A392G showed an increase in
kcat,NAD and a small decrease in the
Km,NAD, with an overall increase in the
kcat,NAD/Km,NAD of 3-fold in relation to the wild-type enzyme. This result indicates that the methyl group of Ala392, which occupies more space
than the corresponding Gly residues, may modify the local polypeptide
conformation in a way that allows a better interaction of the enzyme
with NADP. On the other hand, a Gly residue at this site permits a
better catalytic efficiency when using NAD as cofactor, and points out
this amino acid, among others, as one of the sites that may be
implicated in determining the coenzyme specificity. Earlier protein
engineering of glutathione reductase have indicated an equivalent Ala
(Ala183) as a residue displaying the preference of this
enzyme for NADP, although this substitution was only included in a
multiple mutant but was not studied alone (32).
On the other hand, substitution of Ala387 by Gly provoked a
great decrease in kcat and a moderate increase
in Km (for both NADP and malate), rendering an
enzyme with a 230-fold decrease in
kcat/Km,NADP
(Table I). No NAD-dependent activity could be
measured for the A387G mutant probably because of an overall decrease
in the catalytic efficiency of the mutated enzyme. In this case, the
methyl group of Ala387 seems to be critical for a proper
structure of the active site for catalysis, but not for binding the
substrates. This result is in accord with the high degree of
conservation of an Ala residue in this position in all NAD(P)-ME, which
is replaced only by hydrophobic residues in different NAD(P)-binding
enzymes (30). In this way, a Gly residue at this position may interrupt
the secondary structure (the The last mutant analyzed in the present paper, R237L, changes a
positive charge (Arg residue) near site II by a noncharged residue. The
binding of NADP and malate are both affected (Table I), which is in
accord to the three-dimensional structure obtained for this enzyme that
shows the proximity of this residue to both of these negatively charged
substrates (Fig. 4B). Chemical modifications of maize
NADP-ME have indicated the presence of an essential Arg residue in this
enzyme, which was suggested to play a role in the binding of malate
(33). The crystal structure of the closed form of human mitochondrial
NAD-ME in the presence of substrates has also indicated that
Arg165 (that corresponds to Arg237 of maize
NADP-ME) interacts with both the C1 carboxylate group in
oxalate (used as analog of malate) and with the phosphate group of NAD
(34). Nevertheless, the crystal structure of NAD-ME from A. suum has revealed that the equivalent residue in the enzyme from
this organism (Arg181) does not contribute to NAD binding
and, instead, forms a salt bridge with negatively charged amino acids
(12). In this way, the results obtained in the present paper are more
in accord to the structure of the human mitochondrial enzyme than the
A. suum enzyme, and indicates an important role of this Arg
in the interaction to both NADP and malate.
On the other hand, the dramatic decrease in kcat
for the maize R237L NADP-ME mutant (Table I), suggests that this
residue may also be involved in the catalytic reaction, for example,
acting as a general base for extracting a proton of the C2
hydroxyl of malate or in the protonation of the enolpyruvate
intermediate. A similar decrease in kcat for the
K162A mutant of pigeon liver NADP-ME was observed (35) and thus, this
residue (which corresponds to Lys255 of maize NADP-ME) was
suggested as one of the critical catalytic residues, playing a role as
a general base. In this way, we must conclude that Arg237
in maize NADP-ME may be, not only involved in substrate binding, but
also directly implicated in catalysis. Alternatively, the side chain
and/or charge of this residue may allow other residues (as for example,
Lys255) to be positioned to serve as catalytic base and/or
help to orientate substrates for the catalytic steps. Further studies
mutating the Lys255 residue will elucidate its importance
in the catalytic mechanism of maize C4 NADP-ME.
In conclusion, the present results provide a great advance in the study
of C4 NADP-ME. The use of this recombinant enzyme for
future mutagenesis and crystal structure studies will help to identify
the amino acid residues and/or domains responsible for the
C4-specific properties of this enzyme in relation to other non-C4 NADP-ME and get insight in the evolutionary process
leading from C3 to C4 metabolism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ZAP (Stratagene) cDNA library
from maize leaves, using the procedures described in Sambrook et
al. (13). Positive clones with the estimated complete length of
NADP-ME (~2 kilobase pairs) were identified by KpnI
digestion of the purified DNA from the positive phages by Southern blot
(13). The corresponding plasmids (pBluescript SK) were excised from the
selected phages using the helper phage ExAssist and E. coli
XLOLR strain (Stratagene). The insert of one of the plasmids (pBS-ME)
selected by restriction enzyme analysis was completely sequenced (DNA
Sequencing Facility of Maine University) and corresponded to the
complete cDNA of the C4 NADP-ME from maize (Ref. 14,
GenBankTM accession number J05130).
-mercaptoethanol).
80 °C in buffer B (with 50% glycerol) for further
studies. Protein concentration was determined by the method of
Sedmak and Grossberg (18) using bovine serum albumin as standard.
) was obtained by the
equation,
in which 111.42 was used as MMRW (the
mean amino acid residue weight), d is the cell path in cm,
and c is the concentration of the protein in mg/ml.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain. In this way, total protein extracts obtained from saturated cultures of DH5
E. coli transformed with
pBS-ME were analyzed by Western blot using anti-maize NADP-ME-purified antibodies (15). The results obtained (Fig.
1A) indicated the presence of
an immunoreactive band of 69 kDa present only in E. coli
transformed with pBS-ME. This band was found in extracts induced or not
with isopropyl-1-thio-
-D-galactopyranoside (Fig. 1A), which may be because of the high copy number of this
plasmid. The molecular mass of this immunoreactive band corresponded to the expected mass of the fusion protein as follows: mature NADP-ME (62 kDa) plus the NADP-ME transit peptide and a sequence codified by the
vector. In all the samples analyzed, a second immunoreactive band of 60 kDa was also observed (Fig. 1A), which may correspond to an
endogenous NADP-ME form of E. coli (24).
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Fig. 1.
A, Western blot of protein extracts from
E. coli transformed with pBS-ME. Lane 1, 10 µg
of protein crude extract obtained from maize leaves; lanes 2 and 3, 10 µg of protein from overnight cultures of
DH5 (pBS-ME) grown in the presence or absence of
isopropyl-1-thio-
-D-galactopyranoside, respectively; and
lane 4, 10 µg of protein from overnight cultures of
DH5
(pBS) E. coli strain. B and C,
SDS-PAGE (B) and Western blot (C) of protein
extracts from E. coli transformed with pET32-ME.
B, lane 1, molecular mass markers; C,
lane 1, 20 µg of protein crude extract obtained from maize
leaves; lane 2, 10 µg of protein from overnight cultures
of BL21(pBS-ME); lanes 3 and 4, 10 µg of
protein of the insoluble (3) and soluble (4)
fraction from overnight cultures of BL21(pBS-ME) after
isopropyl-1-thio-
-D-galactopyranoside induction for
5 h. The calculated molecular masses of the immunoreactive bands
are indicated. In maize leaf crude extracts, two immunoreactive bands
corresponding to the photosynthetic (62 kDa) and nonphotosynthetic (72 kDa) NADP-ME isoforms are present (15, 25). Purified antibodies against
maize C4 NADP-ME were used (15, 25).
E. coli cultures transformed with pBS-ME. In this case, DH5
E. coli
transformed with pBS-ME presented more than 10 times more NADP-ME
activity than DH5
E. coli not transformed with the
plasmid (typically 2.9 versus 0.22 IU/mg of the
untransformed strain). The induction of the transformed bacteria with
isopropyl-1-thio-
-D-galactopyranoside resulted in less
NADP-ME activity measurements (typically nearly 1 IU/mg), which may
indicate that overexpression of this fusion protein of NADP-ME may be
detrimental to the bacteria.
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Fig. 2.
SDS-PAGE (A), Western blot
(B), and native IEF revealed for activity
(C) of the recombinant fusion NADP-ME obtained in BL21
E. coli before and after enterokinase digestion.
Lane 1, molecular mass marker; lanes 2 and
3, 10 µg (A), 3 µg (B) and 0.3 mIU (C) of recombinant NADP-ME before (2)
and after (3) enterokinase digestion; lane 4, 20 µg (B) and 0.3 mIU (C) of crude protein extract
obtained from maize leaves. The calculated molecular masses of the
immunoreactive bands and the calculated isoelectric point
(Ip) of the active bands are indicated.
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Fig. 3.
CD (A) and IF emission
(B) spectra of wild-type and mutated maize
C4 NADP-ME. A, CD spectra of wild-type ( )
and Gsite2V (-··-); Gsite5V (- -); A387G (- - -); A392G (-·-),
and R237L (···) mutants of NADP-ME. Five repetitive scans from
200 to 250 nm were performed using 0.1 mg/ml of each enzyme.
B, IF emission spectra of wild-type (
) and Gsite2V
(-··-); Gsite5V (- -); A387G (- - -); A392G (-·-), and R237L
(···) mutants of NADP-ME. Each enzyme was used at a concentration
of 0.02 mg/ml. The spectra were corrected for the buffer absorption and
the Raman spectrum of water.
Kinetic parameters of the recombinant wild-type maize C4
NADP-ME and site-directed mutants analyzed in the present paper
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Fig. 4.
Three-dimensional model view of the maize
chloroplastic C4 NADP-ME corresponding to the active site
of the enzyme. The predicted model was obtained using human
mitochondrial NAD-ME (10) and the pigeon cytosolic NADP-ME (11) as
templates. The figure was created by using Swiss PDB Viewer version
3.7. A, Gly residues 240, 242, and 245 at site II and Gly
residues 383, 385, and 388 at site V are indicated, along with NADP,
Mn2+, and oxalate. This last compound was used as an analog
of malate in the crystallographic studies of pigeon NADP-ME (11).
B, a rotated view of the structure is shown indicating
Arg237 at site II and Ala387 and
Ala392 at site V, residues mutated in the present
paper.
-fold, as previously indicated for residues
311GAGEAA316 of the human mitochondrial NAD-ME
in domain C (10). On the other hand, contrary to expectations from the
sequence analysis of site II (which includes residues
240GLGDLG245 of the maize NADP-ME), this motif
is not involved in a typical Rossman
-fold (Fig.
4A).
-fold and it was previously suggested that Ala residues at this site are diagnostic for NADP specificity over NAD (29, 30), two
mutants were designed to replace Ala residues by Gly, at positions 392 or 387 (A392G or A387G, respectively). In the third mutant studied, an
Arg residue near site II was replaced by Leu (R237L) to verify the
importance of a positive charge residue at this site in binding
negatively charged substrates as NADP and/or malate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase protein, presented malic
enzyme activity and restored the growth on malate of an E. coli mutant, indicating that the fusion protein obtained could
successfully assemble as an active enzyme into the bacteria.
B strand (8, 9). The
first Gly allows the tight turn of the main chain, the second allows a
close contact to the diphosphate of NAD(P), and the third is important for the close packing of the secondary structure. Taking into account
that any side chain in these positions would protrude and disrupt the
cofactor binding, we decided to alternatively disrupt the two
dinucleotide-binding signature motifs in maize NADP-ME (conserved sites
II and V of plant NADP-MEs, see Ref. 1) by changing the Gly residues by
Val, to analyze their relative importance in the cofactor binding. Both
mutants obtained (Gsite2V and Gsite5V), although successfully expressed
and purified, resulted totally inactive, which may indicate that both
sites are essential for activity or, alternatively, that the changes
introduced modify the overall structure of the enzyme rendering
abortive mutants. To test this last possibility, we performed CD and IF
spectra of both proteins (Fig. 3), which indicated that no major
structural changes, in relation to the wild-type enzyme, have occurred
as a result of these mutations.
-fold), modifying the structure
of the active site in a way that the catalytic residues are no longer
well positioned for catalysis. Regarding the coenzyme specificity of
NADP versus NAD, it would be of interest to study the effect
of a change of Gly388 by Ala, as Ala residues are found at
equivalent positions in animal NAD(P)-MEs but Gly residues in plant
NADP-MEs, suggesting that NAD(P)-MEs violate this rule for cofactor
specificity. Nevertheless, this rule was confirmed by protein
engineering of glutathione reductase, where the change of
Ala179 by Gly at an equivalent position in this enzyme,
both in single as well as in multiple substitutions, provoked dramatic
changes associated with NAD preference over NADP (32).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. T. Nelson (Yale University) for providing the partial clone for the maize NADP-ME and Dr. A. Vila (IBR, Rosario, Argentina) for kindly facilitating the use of the CD instrument.
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FOOTNOTES |
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* This work was supported in part by Agencia Nacional de Promoción Científica y Tecnológica Grant BID 802/OC-AR PICT 1-03397, Argentina, and Fundación Antorchas Project 13887/1, Argentina.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.
Fellow of the Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET).
§ Members of the Researcher Career of CONICET.
¶ To whom correspondence should be addressed. Tel.: 54-341-4371955; Fax: 54-341-4370044; E-mail: candreo@fbioyf.unr.edu.ar.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M212530200
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ABBREVIATIONS |
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The abbreviations used are: ME, malic enzyme; Mes, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Mops, 4-morpholinepropanesulfonic acid; IF, intrinsic fluorescence.
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1. | Drincovich, M. F., Casati, P., and Andreo, C. S. (2001) FEBS Lett. 290, 1-6[CrossRef] |
2. | Edwards, G. E., and Walker, D. A. (1983) C3, C4: Mechanism and Cellular Environmental Regulation of Photosynthesis , Blackwell Scientific Publications, Oxford |
3. | Takeuchi, Y., Akagi, H., Kasamura, N., Osumi, M., and Honda, H. (2000) Planta (Heidelberg) 211, 265-274[CrossRef] |
4. | Edwards, G. E., and Andreo, C. S. (1992) Phytochemistry 31, 1845-1857[CrossRef][Medline] [Order article via Infotrieve] |
5. | Sheen, J. (1999) Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 187-217[CrossRef] |
6. | Tausta, S. L., Coyle, H. M., Rothermel, B., Stiefel, V., and Nelson, T. (2002) Plant Mol. Biol. 50, 635-652[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Lai, L. B.,
Wang, L.,
and Nelson, T. M.
(2002)
Plant Physiol.
128,
125-139 |
8. | Rossman, M. G., Liljas, A., Brändén, C. I., and Banaszak, L. J. (1975) The Enzymes , 3rd Ed., Vol. 11 , pp. 61-102, Academic Press, New York |
9. |
Bellamacina, C. R.
(1996)
FASEB J.
10,
1257-1269 |
10. | Xu, Y., Bhargava, G., Wu, H., Loeber, G., and Tong, L. (1999) Structure 7, 877-889[Medline] [Order article via Infotrieve] |
11. |
Yang, Z.,
Zhang, H.,
Hung, H.-H.,
Kuo, C.-C.,
Tsai, L.-C.,
Yuan, H. S.,
Chou, W.-Y.,
Chang, G.-G.,
and Tong, L.
(2002)
Protein Sci.
11,
332-341 |
12. | Coleman, D. E., Jagannatha Rao, G. S., Goldsmith, E. J., Cook, P. F., and Harris, B. G. (2002) Biochemistry 41, 6928-6938[CrossRef][Medline] [Order article via Infotrieve] |
13. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
14. |
Rothermel, B. A.,
and Nelson, T.
(1989)
J. Biol. Chem.
264,
19587-19592 |
15. | Maurino, V. G., Drincovich, M. F., and Andreo, C. S. (1996) Biochem. Mol. Biol. Int. 38, 239-250[Medline] [Order article via Infotrieve] |
16. | Hansen, E. J., and Juni, E. (1975) Biochem. Biophys. Res. Commun. 65, 559-566[Medline] [Order article via Infotrieve] |
17. | Penefsky, H. (1977) J. Biol. Chem. 252, 2891-2899[Abstract] |
18. | Sedmak, J. J., and Grossberg, S. E. (1977) Anal. Biochem. 79, 544-552[Medline] [Order article via Infotrieve] |
19. | Mikaelian, I., and Sergeant, A. (1992) Nucleic Acids Res. 20, 376[Medline] [Order article via Infotrieve] |
20. | Grover, S. D., Canellas, P. F., and Wedding, R. T. (1981) Arch. Biochem. Biophys. 209, 396-407[Medline] [Order article via Infotrieve] |
21. | Ashton, A. R. (1997) Arch. Biochem. Biophys. 345, 251-258[CrossRef][Medline] [Order article via Infotrieve] |
22. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
23. | Burnette, W. N. (1981) Anal. Biochem. 112, 195-203[Medline] [Order article via Infotrieve] |
24. | Spina, J., Bright, H., and Robinson, J. (1970) Biochemistry 9, 3794-3801 |
25. | Maurino, V. G., Drincovich, M. F., Casati, P., Andreo, C. S., Ku, M. S. B., Gupta, S. K., Edwards, G. E., and Franceschi, V. R. (1997) J. Exp. Bot. 48, 799-811[Abstract] |
26. | Spampinato, C. P., Casati, P., and Andreo, C. S. (1998) Biochim. Biophys. Acta 1383, 245-252[Medline] [Order article via Infotrieve] |
27. | Drincovich, M. F., and Andreo, C. S. (1995) Biochem. Mol. Biol. Int. 36, 1287-1297[Medline] [Order article via Infotrieve] |
28. | Drincovich, M. F., Iglesias, A. A., and Andreo, C. S. (1991) Physiol. Plant. 81, 462-466[CrossRef] |
29. | Wierenga, R. K., de Maever, M. C., and Hol, W. G. J. (1985) Biochemistry 24, 1346-1357 |
30. | Hanukoglu, I., and Gutfinger, T. (1989) Eur J. Biochem. 180, 479-484[Abstract] |
31. | Hatch, M. D., and Mau, S. L. M. (1977) Arch. Biochem. Biophys. 179, 361-369[Medline] [Order article via Infotrieve] |
32. | Scrutton, N. S., Berry, A., and Perham, R. N. (1990) Nature 343, 38-43[CrossRef][Medline] [Order article via Infotrieve] |
33. | Rao, S. R., Kamath, B. G., and Bhagwat, A. N. (1991) Phytochemistry 30, 431-435[CrossRef] |
34. | Yang, Z., Floyd, D. L., Loeber, G., and Tong, L. (2000) Nat. Struct. Biol. 7, 251-257[CrossRef][Medline] [Order article via Infotrieve] |
35. | Kuo, C.-C., Tsai, L.-C., Chin, T.-Y., Chang, G.-G., and Chou, W.-Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825[CrossRef][Medline] [Order article via Infotrieve] |