Plant Molecular Biology 55: 97–107, 2004. Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Maize recombinant non-C4 NADP-malic enzyme: A novel dimeric malic enzyme with high specific activity Mariana Saigo1, Federico P. Bologna1, Vero´nica G. Maurino2, Enrique Detarsio1, Carlos S. Andreo1,* and Marı´ a F. Drincovich1 1
Centro de Estudios Fotosinte´ticos y Bioquı´micos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina (*author for correspondence; e-mail
[email protected]); 2Botanisches Institut der Universita¨t zu Ko¨ln, Lehrstuhl II, Gyrhofstrasse 15 50931, Cologne, Germany Received 1 April 2004; Accepted in revised form 28 May 2004
Key words: C4 evolution, NADP-malic enzyme, non-photosynthetic, oligomeric state, prokariotic expression, Zea mays
Abstract Among the different isoforms of NADP-malic enzyme (NADP-ME) involved in a wide range of metabolic pathways in plants, the NADP-ME that participates in C4-photosynthesis is the most studied. In the present work, the expression in E. coli of a cDNA encoding for a maize non-photosynthetic NADP-ME is presented. The recombinant NADP-ME thus obtained presents kinetic and structural properties different from the enzyme previously purified from etiolated leaves and roots. Moreover, the recombinant nonphotosynthetic NADP-ME presents very high intrinsic NADP-ME activity, which is unexpected for a nonC4 NADP-ME. Using antibodies against this recombinant enzyme, an immunoreactive band of 66 kDa is detected in different maize tissues indicating that the 66 kDa-NADP-ME is in fact a protein expressed in vivo. The recombinant NADP-ME assembles as a dimer, although the results obtained indicate that a higher molecular mass oligomeric state of the enzyme is found in maize roots in vivo. In this way, maize presents at least three NADP-ME isoforms: a 72 kDa constitutive form (previously characterized); the novel non-photosynthetic 66 kDa isoform characterized in this work (which is the product of the ZmChlMe2 gene and the likely precursor to the evolution of the photosynthetic C4 NADP-ME) and the 62 kDa isoform (implicated in C4 photosynthesis). The contribution of the present work anticipates further studies concerning the equilibrium between the oligomeric states of the NADP-ME isoforms and the evolution towards the C4 isoenzyme in maize. Introduction NADP-malic enzyme (NADP-ME; EC 1.1.1.40) catalyses the oxidative decarboxylation of malate in the presence of a divalent metal ion. The products of this reaction – pyruvate, CO2 and NADPH – are used in a wide range of metabolic pathways as a source of carbon and reductive power. Thus, distinct isoforms of NADP-ME are expressed in both prokaryotic and eukaryotic organisms, where they play diverse metabolic roles (Chang and Tong, 2003). Animal mitochondrial and cytosolic NADP-MEs have been extensively
studied in relation to lipogenesis and glutaminolysis, and their crystal structures have been recently solved (Xu et al., 1999; Yang et al., 2002). In plants, plastidic and cytosolic isoforms of NADP-ME have been detected (Edwards and Andreo, 1992; Drincovich et al., 2001). In particular, a photosynthetic NADP-ME is expressed in C4 plants, which represents a unique and specialized form of malic enzyme with particular kinetic and regulatory properties (Drincovich et al., 2001). This enzyme is involved in the CO2 concentrating mechanism that increases the photosynthetic yield of NADP-ME C4 plants. It is
98 exclusively expressed in bundle sheath chloroplasts, which present rudimentary grana or are completely agranal probably due to the high level expression of this enzyme (Takeuchi et al., 1998). Apart from this photosynthetic NADP-ME, C4 plants have non-photosynthetic NADP-ME isoforms, which are found in the cytosol (Lai et al., 2002a) and/or plastids (Maurino et al., 1997; Drincovich et al., 1998; Tausta et al., 2002; Lai et al., 2002b). These non-photosynthetic isoforms represent ancestors of the C4 pathway-specific enzyme, which evolved by acquiring elements to make the enzyme suitable to participate in the C4 fixation mechanism – such as high-level expression in bundle sheath chloroplasts and up-regulation by light. In this way, the characterization of these ancestral isoforms are of great importance to shed light on the mechanisms that were involved towards the evolution of the C4 isoform. In maize, the plastidic non-photosynthetic NADP-ME seems to be encoded by two nearly identical genes called ZmChlMe2A and ZmChlMe2B – to distinguish from the one encoding the photosynthetic enzyme (ZmChlMe1) (Tausta et al., 2002). This plastidic NADP-ME represents the more recent and direct ancestor of the photosynthetic C4 enzyme and presents a constitutive pattern of expression in maize (Tausta et al., 2002). It was postulated to be involved in providing carbon and reducing power for different metabolic pathways in non-photosynthetic plastids, as well as implicated in plant defence responses (Maurino et al., 2001; Tausta et al., 2002). In this paper, we expressed a cDNA encoding a plastidic non-photosynthetic NADP-ME in E. coli. The kinetic and structural characteristics of the recombinant NADP-ME thus obtained correspond to a unique NADP-ME, different from the enzyme previously purified from etiolated leaves and roots (Maurino et al., 1996, 2001). This led us to conclude that this enzyme represents an isoform of NADP-ME that has not been yet characterized.
Materials and methods Plant material and protein crude extracts preparation Crude extracts from mature tissues were prepared from plants grown in the field during the summer
for 6–8 weeks (typically 30 °C during the day, 25 °C at night). To prepare crude extracts from seedlings, maize seeds were sown in a mixture of soil and vermiculite and grown for 2 weeks in greenhouse at the same temperature conditions. Maize inbred AX882 (Nidera) was used for all the experiments. Protein crude extracts from different maize tissues were prepared as previously indicated (Maurino et al., 1997). Isolation of non photosynthetic maize NADP-ME cDNA by RT-PCR Total RNA from maize leaves and roots was isolated from 100 mg of tissue using the Trizol reagent (Gibco-BRL). Two microgram total RNA was transcribed using SuperScript II Reverse Transcriptase (Invitrogen) and used directly for RT-PCR. The primers used were: 50 EMR (50 TCTTCTCCATCCGCCGAGCT-30 ) and 30 EMR (50 -ACTACCGGTAGTTACGGTAGACAG-30 ). First-strand synthesis products were amplified by PCR using the Expand High Fidelity PCR System (Roche). The amplified products were cloned into pGEMT-Easy (Promega) and sequenced using the PRISM fluorescent dye-terminator system (Applied Biosystems). The resulting nucleotide sequence was submitted to Genbank (accession no. AY315822). Construction of an expression vector for the mature NADP-ME from maize root A PCR reaction, using as template the plasmid obtained from the RT-PCR using root samples (pGEMT-RME), was performed to create an expression vector for the mature maize root NADP-ME protein. Oligonucleotides primers 5EcoRVRME (50 -TCGGATATCCAGCAGCGA AGGT-30 ) and 3XhoIRME (50 -TCCCTCGAGC TACCGGTAGTTACG-30 ) were used. The cDNA amplified was designed to begin at the first codon of the mature maize root NADP-ME after the transit peptide cleavage site – predicted by both ChloroP V1.1 Prediction Program (Emanuelsson et al., 1999) and TargetP Server v1.01 (Emanuelsson et al., 2000). The primers also introduced unique EcoRV and XhoI sites at the 50 and 30 ends of the NADP-ME insert, respectively, in order to easily subclone the PCR fragment into the pET32 expression vector
99 (Novagen). The construction obtained (pETmRME) was designed in such a way that, after enterokinase digestion of the recombinant fusion protein, only five extra aminoacids are introduced at the N-terminus of the mature root NADP-ME. The insert in the expression vector obtained was sequenced to avoid errors due to PCR and subcloning procedures. Expression and purification of recombinant maize root NADP-ME The pET-mRME expression vector contained the mature maize root NADP-ME cDNA fused to a hexahistidine tag (His-tag) in order to facilitate purification of the expressed fusion protein by a nickel-containing His-Bind column (Novagen). The induction and purification of the fusion protein was carried out as previously described for the photosynthetic NADP-ME (Detarsio et al., 2003). The fusion enzyme was then concentrated on Centricon YM-50 (Amicon) using Buffer B (50 mM Tris-HCl pH 8.0, 10 mM MgCl2 and 10% (v/v) glycerol). Purified fusion NADP-ME protein was then incubated with enterokinase (1:100) in buffer B at 15 °C for 1 h in order to remove the N-terminus codified by the expression vector. The addition of 1 mM PMSF in the digestion medium was necessary to obtain only one protein product after incubation. The protein was further purified using an affinity Affi Gel Blue column (BioRad) followed by a second Niquel-NTA column. The purified enzyme was stored at )80 °C in buffer B (with 50% glycerol) for further studies. Preparation of 66 kDa-NADP-ME antiserum Polyclonal antibodies against the 66 kDa recombinant NADP-ME were obtained by immunization of rabbits with 200 lg of the purified protein in four subcutaneous injections of 50 lg at 15 days intervals. The antibodies against the recombinant maize root 66 kDa NADP-ME were further purified from the crude antiserum (Plaxton, 1989). The same procedure was used to obtain antibodies against the recombinant photosynthetic NADP-ME previously expressed in E. coli (Detarsio et al., 2003).
NADP-ME activity assays NADP-ME activity was determined spectrophotometrically as previously indicated (Maurino et al., 1996). 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 from triplicate determinations and adjusted to non-lineal regression. The optimal pH of the recombinant NADP-ME reaction was determined as indicated (Maurino et al., 1996). Malic enzyme activity in the presence of NAD was measured as previously described for the recombinant photosynthetic NADP-ME (Detarsio et al., 2003). Gel electrophoresis SDS-PAGE was performed in 8% or 10% (w/v) polyacrylamide gels according to Laemmli (1970). Proteins were visualized with Coomassie blue or electroblotted onto a nitrocellulose membrane for immunoblotting. Affinity-purified antibodies against the purified maize photosynthetic NADPME (Maurino et al., 1996), along with antibodies obtained in the present work, were used for detection (1:100). Bound antibodies were detected by incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG according to the manufacturer’s instructions (Sigma). Alkaline phosphatase activity was detected colorimetrically or, alternatively, by using a chemiluminescent kit (Immun-Star, BioRad). Native PAGE was performed employing a 6% (w/v) acrylamide separating gel. Electrophoresis was run at 150 V and 10 °C. Gels were analyzed by Western blotting or assayed for malic enzyme activity (Maurino et al., 1997). Native molecular mass estimation The molecular mass of the recombinant native NADP-ME protein was evaluated by gel-filtration chromatography on a HPLC system (Waters Associates) with a BioSep-SEC-S300 column (Phenomenex) or, alternatively, with a Sephacryl S300 (Sigma) column connected to an FPLC-system (Pharmacia). The columns were equilibrated and used as previously described (Detarsio et al., 2003).
100 Results RT-PCR cloning of a full-length cDNA from maize non-photosynthetic NADP-ME Two nearly identical cDNAs (98%), ZmChlMe2A (U39958) and ZmChlMe2B (AY040616), were isolated by RT-PCR from maize roots (Tausta et al., 2002). In order to characterize the nonphotosynthetic NADP-ME expressed in maize roots, the corresponding cDNA was amplified by RT-PCR using primers designed according to ZmChlMe2A and ZmChlMe2B sequences. We obtained independent cDNAs from maize roots (AY315822) with 99% identical sequence to ZmChlMe2A, except for the absence of an extra amino terminal region (see discussion), and 98% identical sequence to ZmChlMe2B. We used this isolated cDNA to express the mature maize root NADP-ME in a prokaryotic system and study its properties. An identical cDNA was also isolated from maize green leaves confirming the postulated constitutive pattern of expression of this nonphotosynthetic NADP-ME (Tausta et al., 2002). The alignment among the predicted aminoacid sequences of the non-photosynthetic NADP-ME cDNAs isolated from maize root in Tausta et al. (2002) and in the present work is presented in Figure 1. Expression and purification of recombinant mature non-photosynthetic NADP-ME from maize The full-length coding region of the isolated nonphotosynthetic NADP-ME cDNA encodes a protein of 644 aminoacids with a predicted molecular mass of 70 766 (http://us.expasy.org/). The sequence possesses an N-terminal extension which is predicted to be a plastidial-targeted transit peptide. The precise location of the processing site is still not known but it was predicted to be at position 39 (Emanuelsson et al., 1999, 2000). Without this transit peptide, the mature NADP-ME is predicted to have a molecular mass of 66 654 and a denatured isoelectric point of 6.07. After removing the region encoding for the predicted plastidic transit peptide, the sequence codifying for the mature non-photosynthetic NADP-ME was cloned in the pET32 expression vector (pET-mRME). E. coli strain BL21 transformed with pET-mRME over-expressed a protein
of 83 kDa after induction with IPTG or lactose. The calculated molecular mass of this protein corresponds to the expected molecular mass of the fusion protein as follows: mature non-photosynthetic maize NADP-ME (66 kDa, see above) plus 17 kDa codified by the expression vector. The 83 kDa fusion protein obtained was purified by a His-Bind affinity column (Figure 2A). After enterokinase digestion of this protein, a 66 kDa protein was obtained (Figure 2A). The 66 kDa protein contains aminoacids encoded by the vector sequence (AlaMetGlyTyrPro) followed by aminoacid residues of the mature non-photosynthetic NADP-ME (AlaAlaLysVal). The 66 kDa value corresponds to the molecular mass of the mature non-photosynthetic NADP-ME predicted from its sequence (see above). This protein reacted with anti-maize photosynthetic NADP-ME purified antibodies (Maurino et al., 1996; Figure 2B), although a 66 kDa band was not detected with these antibodies using maize crude extracts (Figure 2B). Expression of the 66 kDa-NADP-ME in maize tissues In vivo expression of the recombinant 66 kDa NADP-ME in different maize tissues was analysed by Western blot. Studies were performed using polyclonal antibodies previously obtained against the photosynthetic maize NADP-ME purified from leaves (Maurino et al., 1996) and a batch of antibodies against the recombinant plastidic nonphotosynthetic NADP-ME obtained in the present work. When incubated with antibodies against the purified enzyme from maize green leaves, two major NADP-ME immunoreactive bands of 62 and 72 kDa are found in denaturing gels in different maize tissues (Figure 3A). The 62 kDa band is only found in photosynthetic tissues (lanes 3, 6 and 7, Figure 3A) and represents the C4 NADPME found in bundle sheath chloroplasts (Maurino et al., 1997; Detarsio et al., 2003). The 72 kDa immunoreactive band is found in all the tissues tested (roots, leaves, husk and central vein) being the only band detected in non-photosynthetic tissues (lanes 4, 5 and 8, Figure 3A). This isoform was suggested to be a plastidic non-photosynthetic NADP-ME (Maurino et al., 1996, 1997, 2001) and was also detected with different batches of antibodies against NADP-ME (Tausta et al., 2002).
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Figure 1. Comparison of the predicted aminoacid sequences of different non-photosynthetic NADP-ME cDNAs isolated from maize root. The sequences predicted from ZmChlMe2A (U39958, GenPep AAD10504), ZmChlMe2B (AY040616, GenPep AAK91502) and the sequence obtained in the present study (AY315822) were aligned using CLUSTAL W (1.82).
On the other hand, when the same tissue samples were probed with antibodies against the 66 kDa recombinant non-photosynthetic
NADP-ME, two immunoreactive bands were found: the 62 kDa NADP-ME and a second immunoreactive band of 66 kDa (Figure 3B). This
102 tive band of the same molecular mass of the recombinant NADP-ME obtained in the present work is now detected. This immunoreactive band of 66 kDa is clearly found in roots from seedlings and mature plants, as well as in husks (lanes 4, 8 and 5, respectively; Figure 3B), being the only immunoreactive band present in roots. When examining photosynthetic tissues, a band of 66 kDa can also be detected as a faint band near the 62 kDa NADP-ME. This band is more clearly visible in husks and mature leaves (lanes 6 and 7, respectively, Figure 3B) and it was also detectable in seedling leaves depending on the amount of protein loaded. It is also noteworthy that when using antibodies to recombinant photosynthetic NADP-ME (Detarsio et al., 2003) or to recombinant non-photosynthetic NADP-ME the same pattern of bands was obtained in both cases (data not shown). Kinetic characterization of recombinant non-photosynthetic NADP-ME from maize Figure 2. Coomassie stained SDS-PAGE (A) and Western blot (B) of recombinant non-photosynthetic NADP-ME obtained in BL21 E. coli before and after enterokinase digestion. Lane 1: molecular mass marker; lane 2 and 3: 5 lg (A) and 2 lg (B) of recombinant NADP-ME before (2) and after (3) enterokinase digestion; lanes 4 and 5: 20 lg of protein crude extract obtained from maize roots (4) and leaves (5). The calculated molecular masses of the immunoreactive bands are indicated. Antibodies obtained against the purified photosynthetic NADP-ME were used (14).
indicates that the antibodies raised against the recombinant non-photosynthetic NADP-ME still recognize the photosynthetic enzyme (62 kDa) but not the 72 kDa protein. Instead, an immunoreac-
The apparent kinetic parameters of the recombinant 66 kDa non-photosynthetic NADPME obtained in the present work were estimated and compared to the values previously obtained for the purified NADP-ME from maize roots, and for the purified and recombinant maize photosynthetic C4 NADP-ME (Table 1). The recombinant maize non-photosynthetic NADP-ME presented an unexpectedly high kcat for a non-photosynthetic enzyme, being 62-times higher than the value previously obtained for the enzyme purified from maize roots and etiolated leaves (Table 1). Moreover, this kcat value was
Figure 3. Western blot analysis of NADP-ME in different maize tissues. Crude extracts (25 lg) of different maize tissues were incubated with antibodies against purified photosynthetic NADP-ME (A) and recombinant non-photosynthetic NADP-ME (B). Seedling leaves (3), seedling roots (4), husk (5), central vein (6), mature leaves (7) and mature roots (8). Recombinant non-photosynthetic (lane 1, 2 lg) and photosynthetic NADP-ME (lane 2, 2 lg) were used as controls. The calculated molecular masses of the immunoreactive bands are indicated. In lane 3, the central vein, along with approximately 2 mm of leaf tissue was excised and used for protein extraction.
103 Table 1. Kinetic parameters and physical properties of recombinant non-photosynthetic maize NADP-ME. The table compares the properties with other maize NADP-MEs previously characterized: purified non-photosynthetic NADP-ME (Maurino et al., 2001); purified photosynthetic C4 NADP-ME (Drincovich et al., 1991) and recombinant photosynthetic C4 NADP-ME (Detarsio et al., 2003). The indicated values are the average of at least three different determinations with no more than 5% S.D. among them. Properties
Recombinant non-photosynthetic NADP-ME
Purified non-photosynthetic NADP-ME
Recombinant photosynthetic C4 NADP-ME
Purified photosynthetic C4 NADP-ME
Monomer molecular mass Native oligomeric state pH optimum Malate inhibition at pH 7.0 Ki malate (mM) kcat,NADP (s)1) Km NADP (lM) Km malate (mM) kcat,NAD (s)1) Km NAD (mM)
66 kDa Dimer 7.8 No – 105.6a 70.2a 0.42a,c 0.84a 3.6a
72 kDa Tetramer 7.5 No – 1.7a 20.0b 0.68b nd nd
62 kDa Tetramer 8.0 Yes 4.7 201.3a 8.0a 0.23a 13.5a 8.1a
62 kDa Tetramer 8.0 Yes 4.5 30.8a 8.6a 0.19a nd nd
nd, not determined. a Measured at the optimum pH. b At pH 8.0. c S0.5 (nH = 1.2).
closer to that determined for the recombinant photosynthetic NADP-ME, which, in turn, presented a nearly 7-fold higher kcat than the purified enzyme from maize leaves due to partially inactivation of the enzyme during the purification procedure (Detarsio et al., 2003). The recombinant non-photosynthetic NADP-ME presented also a higher optimum pH for activity than the previously purified enzyme from maize roots, although remained lower than the value determined for the photosynthetic enzyme (Table 1). With regards to the Km values for NADP and L-malate, several differences were found in relation to the purified enzyme from maize roots. A 3.5times higher Km for NADP with regards to the previously value measured for the purified enzyme from roots was obtained. This value was also nearly nine times higher than the one obtained for the photosynthetic enzyme (Table 1). On the other hand, the kinetic behaviour estimated with respect to malate was non-hyperbolic, presenting some kind of sigmoidicity (Table 1). No inhibition by L-malate was found at pH 7.0, in agreement with the conclusion that only the photosynthetic NADP-ME is regulated by malate due to lightdependent changes in the chloroplastic stromal pH (Edwards and Andreo, 1992; Drincovich et al., 2001). This result is also consistent with the fact that the C4 NADP-ME tetramer from sugar cane was the oligomeric form mainly affected by malate
inhibition at pH 7.0 (Iglesias and Andreo, 1990), in view of the oligomeric state of the recombinant non-photosynthetic NADP-ME (see below). The recombinant non-photosynthetic NADPME was also active with NAD; although the Km value obtained was 50 times higher while the kcat was nearly 130 times lower than the values obtained in the presence of NADP (Table 1). This result is similar to those obtained with the recombinant photosynthetic NADP-ME (Detarsio et al., 2003), although the non-photosynthetic NADP-ME presents more affinity for NAD. The activity with this substrate is much lower than for its photosynthetic counterpart (Table 1). Quaternary structure of the recombinant non-photosynthetic NADP-ME from maize Activity gels for NADP-ME in crude extracts from maize leaves and roots show the presence of active NADP-ME bands consistent with a tetrameric state of the enzyme in both tissues (lanes 1 and 2, Figure 4A). The same result was previously obtained (Maurino et al., 1996, 2001) and the active bands were attributed to the photosynthetic 62 kDa NADP-ME in leaves and the 72 kDa enzyme in roots. However, as the recombinant non-photosynthetic NADP-ME obtained in the present work presented an unexpected molecular mass by
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Figure 4. Activity gels (A) and Western blots (B) using antibodies against recombinant non-photosynthetic NADP-ME. Lanes 1 and 2, crude extracts from maize leaves and roots, respectively; 3 and 4, recombinant photosynthetic and non-photosynthetic NADP-ME, respectively; 5 and 6, recombinant photosynthetic and non-photosynthetic NADP-ME fused to the Thioredoxin-His-tag (not digested with enterokinase). For (A) between 1 and 2 mU of enzyme were loaded in each case. For (B) 25 lg of protein were loaded in lanes 1 and 2 and 0.5–1.0 lg in lanes 3–6. Western blots were developed using a chemiluminiscent kit. Native molecular mass markers were run in parallel and stained with Coomassie blue (on the left).
denaturing electrophoresis, native electrophoresis was performed using maize root crude extracts in parallel with the recombinant non-photosynthetic NADP-ME. Maize leaf crude extracts and recombinant photosynthetic NADP-ME (Detarsio et al., 2003) were also included as controls. The results obtained show that the recombinant non-photosynthetic NADP-ME migrates with a molecular mass more consistent with a dimer, although the enzyme from maize roots crude extract exists as a tetramer (lane 4 in relation to lane 2, Figure 4A). On the other hand, the recombinant photosynthetic NADP-ME presents the same mobility (consistent with a tetramer) as the enzyme found in maize leaves crude extract (lane 3 in relation to lane 1, Figure 4A). The recombinant non-photosynthetic and photosynthetic NADP-MEs with fused histidine and thioredoxin tags at the amino terminal (not digested with enterokinase) were also suggested to exist as dimer and tetramer, respectively (lanes 5 and 6, Figure 4A). These fusion proteins still contain a 17 kDa peptide unlike the enterokinase-digested enzymes and, thus, their native molecular masses are higher. In this case, although the molecular mass of the non-digested recombinant non-photosynthetic NADP-ME seems to be larger than the expected for a dimer (lane 6, Figure 4A), the tetramer of this protein
should present a molecular mass larger than the undigested recombinant photosynthetic NADPME (Lane 5, Figure 4A). Western blotting analysis of native gels using antiserum raised against the recombinant nonphotosynthetic NADP-ME (Figure 4B) shows that these antibodies recognize all the NADP-ME active bands detected in Figure 3A. It is worth to mention that these antibodies recognize the NADPME found in maize roots crude extracts (lane 2, Figure 4B). In this way, since these antibodies only recognize the 66 kDa immunoreactive band in maize root crude extracts (Figure 3B), this active band is suggested to be made up by the 66 kDa enzyme, solely or in association to other protein. Although a signal consistent with a dimeric state is detected by Western blot when analyzing recombinant photosynthetic NADPME (lane 3, Figure 4B), this form results in an enzymatic species with no detectable activity (lane 3, Figure 4A). This is not the case with the recombinant non-photosynthetic NADP-ME, where the dimer is the enzymatically active form. In this particular case, a second minor band is detected by Western blot (lane 4, Figure 4B). The molecular mass of this band is comparable to that of the enterokinase-undigested enzyme, and probably represents some undigested product still present in the preparation.
105 Activity gels and Western blot analysis of recombinant non-photosynthetic NADP-ME incubated with its substrates and cofactors (NADP, malate and/or Mg2+), resulted also in bands consistent with a dimeric state of the enzyme. Similar results were obtained when incubating the recombinant NADP-ME with maize root protein crude extracts preincubated at 60 °C in order to inactivate the endogenous NADP-ME (data not shown). To confirm its dimeric form, the recombinant non-photosynthetic enzyme was subjected to gel filtration chromatography. A molecular mass of 130 kDa was obtained at both pH 7.0 or 8.0, confirming the results obtained using native electrophoresis. On the other hand, gel filtration chromatography performed with the recombinant photosynthetic NADP-ME, indicated that this enzyme was able to assemble into a tetrameric form at pH 8.0 and as a mixture of tetramer and dimer at pH 7.0.
Discussion In the present work, a maize root NADP-ME cDNA that displays a high degree of identity to the previously isolated NADP-ME sequences was cloned (Figure 1). The most relevant difference is the lack of extra nucleotides in the 50 region in the cDNA we isolated, which results in the absence of 19 aminoacids in the predicted protein. Nevertheless, as this extra region is not found in any other known NADP-ME and, as indicated by the authors, this region could be a result of alternative or misplicing events (Tausta et al., 2002), we conclude that the protein expressed and characterized in vitro in this study represents a plastidic non-photosynthetic NADP-ME isoform from ZmChlMe2. The other differences, apart from the additional 50 region, between both ZmChlMe2A and ZmChlMe2B in relation to the cDNA isolated in the present work could be due to the use of different inbred maize lines. In the present work, we were not able to isolate other cDNAs with significant differences at the nucleotide level that could be the result of the expression of different genes. In this way, we conclude that the enzyme characterized represents a plastidic non-photosynthetic NADP-ME with a constitutive pattern of expression in maize. With
regard to the plastidic localization of this nonphotosynthetic NADP-ME, the functionality of the transit peptide of ZmChlMe2A was studied by a chloroplast import assay (Tausta et al., 2002). A precursor of 78 kDa and a processed protein of 68 kDa after chloroplast import was obtained, which is in accordance to the 66 kDa of the recombinant mature NADP-ME obtained in the present work, which lacks 19 aminoacids from the amino terminal region. The cDNAs isolated from maize roots should encode a mature protein with a smaller molecular mass than the enzyme previously purified from maize etiolated leaves and roots (Maurino et al., 1996, 2001). In correlation with this, we obtained a product of 66 kDa as the processed enzyme derived from the maize root NADP-ME cDNA expressed in E. coli (after removing the predicted plastidic transit peptide; Figure 2), although a band of this molecular mass was not detected in maize tissues using previously obtained antibodies (Maurino et al., 1996; Figure 3A). Nevertheless, after obtaining antibodies against this recombinant enzyme and probing them against different maize tissues, a 66 kDa protein was detected as a product expressed in vivo (Figure 3B). The pattern of expression of this 66 kDa protein (detected with the antibodies against the recombinant NADP-ME; Figure 3B) correlated with the expression of the 72 kDa NADP-ME in roots (detected with the antibodies against the purified photosynthetic NADP-ME; Figure 3A), although the antibodies against the recombinant 66 kDa enzyme showed no cross-reaction with this 72 kDa protein. Moreover, antibodies against the recombinant photosynthetic NADP-ME (Detarsio et al., 2003) detected also a protein of 66 kDa in vivo, and showed no cross-reaction with the 72 kDa protein. The fact that the antibodies raised against the recombinant NADP-ME, but not those raised against the purified NADP-ME, recognize the 66 kDa protein in vivo, could be due to differential reactivity. They might also interact with different epitopes. In this way, we conclude that the 66 kDa NADP-ME is in fact the product of the ZmChlMe2 cDNA expressed in vivo. Previous reports have also shown a NADP-ME with similar molecular mass in different maize tissues (Tausta et al., 2002) and in different species of Flaveria (Drincovich et al., 1998). A C4 NADPME of 67 kDa was purified from Haloxylum
106 persicum, an unusual tree originally from central Asian deserts (Casati et al., 1999). Native electrophoresis suggested that the recombinant non-photosynthetic NADP-ME obtained in the present work assembles as a dimer (Figure 4), which was confirmed by size exclusion chromatography. This result is in conflict with a NADP-ME of higher molecular mass in maize root crude extracts, which is more in accordance with a tetrameric state (Figure 4). Nevertheless, the antibodies raised against the recombinant 66 kDa NADP-ME recognize this active NADPME from maize roots (Figure 4B), indicating that this enzyme is made up of 66 kDa monomers. Different explanations are consistent with these results. First, the tetramerization of the non-photosynthetic NADP-ME may be due to the action of some metabolite or protein in vivo which, in turn, affects enzyme conformation. Second, the 66 kDa NADP-ME exists as a dimer in vivo, but presents a higher molecular mass due to its association with other proteins. Finally, we cannot rule out the possibility that the 66 kDa NADP-ME expressed in the present work presents in fact some difference(s) with the NADP-ME expressed in vivo, e.g. different transit peptide processing site. The kinetic parameters obtained for the recombinant NADP-ME indicate that the enzyme was totally different from that previously purified from maize etiolated leaves and roots (Table1). The kcat obtained was very high in comparison to values previously obtained for non-photosynthetic enzymes (Drincovich et al., 2001). It is almost 2 times lower than the value obtained for the maize recombinant photosynthetic NADP-ME (Table 1) but between 60 and 100 times higher than values previously obtained for non-photosynthetic enzymes. This high kcat is even more surprising when considering that the enzyme assembles as a dimer in vitro. Previous work has indicated that the dimer and the monomer, although still active, showed markedly less activity than the tetrameric form of the NADP-ME from different sources (Iglesias and Andreo, 1990; Huang and Chang, 1992). After analysing the Km values for the substrates, we conclude it is possible that the higher value obtained in the case of NADP and the sigmoidicity in the case of malate (Table 1) are a consequence of the dimeric state of the enzyme. Previous studies have indicated that the Km for NADP in the tetrameric form is lower than that of
the dimer obtained by dilution of the native tetrameric C4 NADP-ME from sugar cane leaves (Iglesias and Andreo, 1990). To summarize, in the present work we characterized a recombinant dimeric NADP-ME that displays very high intrinsic activity and is totally different from the enzyme previously characterized from maize etiolated leaves and roots. Our results indicate that the number of NADP-ME isoforms found in maize is greater than previously supposed. Although this enzyme seems to present a different oligomeric state in vivo, the recombinant dimeric NADP-ME will be very useful for further studying the characteristics of its different oligomeric states. In this regard, it is intriguing why the recombinant photosynthetic NADP-ME assembles as a tetramer while the recombinant non-photosynthetic enzyme is not able to do so. We speculate that during the evolution to the C4 photosynthetic NADP-ME, changes in some aminoacids have occurred in order to obtain a tetrameric NADP-ME. The advantages for this oligomeric state should be established. It is also worth to mention that the tetrameric state of the animal NADP-MEs, whose crystal structures have been resolved, were found to be a dimer of dimers (Xu et al., 1999; Yang et al., 2002). Identifying the aminoacids that favor one oligomeric state over the other is key to further our understanding of the maize recombinant non-photosynthetic NADP-ME and its relation to other tetrameric NADP-MEs.
Acknowledgements This research was supported by grants from the Agencia Nacional de Promocio´n Cientı´ fica y Tecnolo´gica (PICT 1-03397 and 1-11604, Argentina), Fundacio´n Antorchas (Project N° 13887/1 and N° 14116/24, Argentina) and CONICET (PIP 3029). MFD and CSA are members of the Researcher Career of CONICET and MS and ED are fellows of the same institution. The authors want to thank Dr. Claudio F. Pairoba for carefully reading the manuscript and his suggestions.
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