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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Regulation of Photosynthetic GAPDH Dissected by Mutants¹

Laboratory of Molecular Plant Physiology, Department of Biology, University of Bologna, 40126 Bologna, Italy (F.S., M.Z., P.P., P.T.); and Department of Plant Physiology, Faculty of Biology and Chemistry, University of Osnabrueck, D–49069 Osnabrueck, Germany (N.W., R.S.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants catalyzes an NADPH-consuming reaction, which is part of the Calvin cycle. This reaction is regulated by light via thioredoxins and metabolites, while a minor NADH-dependent activity is constant and constitutive. The major native isozyme is formed by A- and B-subunits in stoichiometric ratio (A₂B₂, A₈B₈), but tetramers of recombinant B-subunits (GapB) display similar regulatory features to A₂B₂-GAPDH. The C-terminal extension (CTE) of B-subunits is essential for thioredoxin-mediated regulation and NAD-induced aggregation to partially inactive oligomers (A₈B₈, B₈). Deletion mutant B(minCTE) is redox insensitive and invariably tetrameric, and chimeric mutant A(plusCTE) acquired redox sensitivity and capacity to aggregate to very large oligomers in presence of NAD. Redox regulation principally affects the turnover number, without significantly changing the affinity for either 1,3-bisphosphoglycerate or NADPH. Mutant R77A of GapB, B(R77A), is down-regulated and mimics the behavior of oxidized GapB under any redox condition, whereas mutant B(E362Q) is constantly up-regulated, resembling reduced GapB. Despite their redox insensitivity, both B(R77A) and B(E362Q) mutants are notably prone to aggregate in presence of NAD. Based on structural data and current functional analysis, a model of GAPDH redox regulation is presented. Formation of a disulfide in the CTE induces a conformational change of the GAPDH with repositioning of the terminal amino acid Glu-362 in the proximity of Arg-77. The latter residue is thus distracted from binding the 2'-phosphate of NADP, with the final effect that the enzyme relaxes to a conformation leading to a slower NADPH-dependent catalytic activity.

At difference from thioredoxins, thioredoxin targets have no consensus sequences, and it is still unclear whether the molecular mechanisms of thioredoxin regulation follow any general rule. Based on crystal structures of reduced/oxidized forms and the kinetic analysis of site-specific mutants, the redox regulations of Fru bisphosphate phosphatase and NADP-malate dehydrogenase were satisfactorily explained at the molecular level and found to be different (Carr et al., 1999; Chiadmi et al., 1999; Johansson et al., 1999). No other thioredoxin-regulated enzyme has been investigated in such depth.

Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a target of thioredoxins in plant chloroplasts (Wolosiuk and Buchanan, 1978; Sparla et al., 2002). Two isoforms of chloroplast GAPDH exist in higher plants, an abundant heteromeric AB-isoform and a minor homotetrameric A₄-isoform, with quite different regulatory properties (Cerff, 1979; Brinkmann et al., 1989; Ferri et al., 1990; Scagliarini et al., 1993). A₄-GAPDH is regulated through the interaction with the small protein CP12, giving a supramolecular complex with phosphoribulokinase (Wedel and Soll, 1998; Graciet et al., 2003). Formation and dissociation of this supramolecular complex contributes to light-dependent modulation of both enzyme activities and hence to the overall regulation of photosynthetic metabolism (Scheibe et al., 2002; Graciet et al., 2004). The crystal structure of spinach (Spinacia oleracea) A₄-GAPDH in complex with either NADP or NAD has recently been solved (Fermani et al., 2001; Falini et al., 2003). It was argued that Arg-77 and Ser-188, by specifically interacting with the 2'-phosphate of NADP, are key residues for coenzyme specificity in this protein (Sparla et al., 2004).

Although AB-GAPDH may also participate in supramolecular complexes with CP12 and phosphoribulokinase (Clasper et al., 1991; Wedel et al., 1997; Scheibe et al., 2002), AB-GAPDH exhibits in addition an autonomous type of regulation (Pupillo and Giuliani Piccari, 1975), which depends on the presence of a C-terminal extension (CTE) characteristic of B-subunits and homologous to the C-terminal end of CP12 (Baalmann et al., 1996; Pohlmeyer et al., 1996). The enzyme activity is modulated through the reversible formation of a disulfide bridge in the CTE between Cys-349 and Cys-358, operated by thioredoxin f in relation to the light/dark cycle (Sparla et al., 2002). Redox regulation of AB-GAPDH selectively affects the turnover number of the NADPH-dependent activity, leaving unchanged the alternative activity with NADH as coenzyme. A comparable decrease of NADPH-dependent activity was recently obtained in recombinant A₄-GAPDH impaired in coenzyme recognition due to the substitution of Ser-188 into Ala. The data thus suggest a link between coenzyme specificity and redox regulation of the GAPDH (Sparla et al., 2004).

With the aim of unraveling the molecular mechanism of thioredoxin regulation in AB-GAPDH, here we report on a series of mutants affected in redox regulation. By integrating information derived from kinetic analysis of these mutants and tridimensional structures of A₄-GAPDH, a molecular model of AB-GAPDH regulation by thioredoxin was derived. Given the similarity between CP12 and CTE, this model provides a molecular basis for a broader understanding of photosynthetic GAPDH regulation including the CP12-dependent regulation of A₄-type isozymes.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Protein Engineering, Heterologous Expression, and Purification of Regulatory Mutants of Photosynthetic GAPDH

Of the two subunits present in the AB-type photosynthetic GAPDH, subunit-B contains the autoinhibitory domain CTE that confers regulatory properties to the whole enzyme. Different site-specific mutants of GAPDH B-subunits, i.e. B(S188A), B(R77A), and B(E362Q), were constructed and expressed in Escherichia coli (Fig. 1). The rationale behind the choice of mutations S188A and R77A in GapB was that both residues are involved in NADP versus NAD recognition in recombinant A₄-GAPDH (Sparla et al., 2004) and, therefore, are possible candidates for NADPH-specific regulation of GapB. Glu-362, the C-terminal amino acid of B-subunits, was mutated into Gln with the aim of decreasing the overall negative charge of the CTE domain. This domain was suggested to make electrostatic interactions with the positively charged S-loop (Asp-181 to Ala-199), which binds NADP via Ser-188 (Fig. 1; Reichert et al., 2000; Sparla et al., 2002). The chimeric mutant A(plusCTE) and deletion mutant B(minCTE) (Baalmann et al., 1996) were also constructed and expressed in E. coli as a further test for the regulatory function of CTE.

View larger version (37K): [in this window] [in a new window]   Figure 1. Amino acid sequence and schematic representation of wild-type and mutant forms studied in this work. A, Pairwise alignment of GapA and GapB subunits of spinach GAPDH. Residues are numbered according to the structure of Bacillus stearothermophilus GAPDH deposited in the Protein Data Bank (Biesecker et al., 1977). Insertion or deletions with respect to B. stearothermophilus GAPDH are underlined. Amino acids identical in both GapA and GapB are on a gray field. Site specific mutations of Arg-77, Ser-188, and Glu-362 are on a black field. Sequences corresponding to the S-loop and the CTE are double underlined, and charged amino acids within these sequences are indicated by symbols + and –. The regulatory disulfide bridge between Cys-349 and Cys-358 (Sparla et al., 2002) is indicated. B, Schematic survey of all GAPDH forms studied in this work. GapA is represented by a gray rectangle, GapB by a white rectangle (positions 1–334), plus a black rectangle corresponding to the CTE (positions 335–362). Site-specific substitutions are reported in bold.

Sensitivity of Regulatory Mutants to the Redox State of Thioredoxin

The regulation of spinach AB-GAPDH activity by thioredoxin f was previously shown to depend on B-subunits and to specifically affect the NADPH-dependent activity (E_m,7.9 –353 ± 11 mV; Fig. 2; Baalmann et al., 1996; Li and Anderson, 1997; Sparla et al., 2002). Redox modulation was not due to a change in affinity for substrate (1,3-bisphosphoglycerate) or coenzyme (NADPH), but rather to a 2.2-fold shift in maximal catalytic activity (Table I; Sparla et al., 2004). As a consequence, redox titrations of enzyme activities measured with standard assay (Fig. 2) approximately indicate specific changes in V_max.

View larger version (26K): [in this window] [in a new window]   Figure 2. Redox titration of eight different wild-type and mutant forms of spinach GAPDH in the presence of spinach thioredoxin f. Activities were measured under standard conditions (see "Materials and Methods") with either NADPH (black circles) or NADH (white circles) as electron donor. All GAPDH forms displayed a maximal catalytic activity with NADPH as coenzyme under reducing conditions in the range of 80 to 130 µmol min–1 mg–1, with the exception of mutant B(R77A) whose activity under these conditions was 46 µmol min–1 mg–1. In the graphs, activities under varying redox conditions have been normalized to the NADPH-activity of each form under fully reducing conditions, which was set to 1 for all GAPDH forms with similar maximal activity (80–130 µmol min–1 mg–1) and set to 0.5 for B(R77A) (46 µmol min–1 mg–1).

The involvement of CTE in the regulatory mechanism was confirmed by the redox insensitivity of B(minCTE) deletion mutant (Fig. 2; Li and Anderson, 1997; Sparla et al., 2002). On the other hand, the newly described chimeric mutant A(plusCTE) gained redox sensitivity as expected (Fig. 2). However, despite the extensive sequence homology between GapA and GapB (CTE excluded), the behavior of mutant A(plusCTE) was different from GapB in that both NADPH- and NADH-dependent activities were found to be similarly redox-modulated (Fig. 2) and decreased upon enzyme oxidation to about 50% of the fully reduced enzyme. Midpoint redox potentials at pH 7.9 were –340 ± 14 mV and –345 ± 18 mV for the NADPH- and the NADH-dependent activities, respectively, slightly less negative than AB-GAPDH and recombinant GapB. The affinity of A(plusCTE) for BPGA [K_m(BPGA) about 20 µM] was as in GapA regardless of the redox state (Table I), that for NADPH was somewhat lower [K_m(NADPH) 65 µM reduced, 43 µM oxidized]. Albeit not identical to GapB, the redox sensitivity of A(plusCTE) clearly shows the capability of recombinant CTE to modulate the activity of nonregulatory GAPDH isoforms.

The site-specific mutant B(S188A) was also characterized by redox sensitivity of both NADPH- and NADH-dependent activities (Fig. 2). Midpoint redox potentials for both reactions were less negative than all other redox-sensitive GAPDH forms (NADPH, E_m,7.9 –329 ± 7 mV; NADH, E_m,7.9 –346 ± 14 mV). Redox modulation of this mutant was associated with a slightly lower affinity for the substrates [K_m(BPGA) about 31 µM; K_m(NADPH) about 46 µM, reducing conditions; Table I].

Crystal structures of recombinant GapA showed that the extra 2'-phosphate group of NADP is stabilized by Arg-77 besides Ser-188 (Sparla et al., 2004). Consistent with this finding, substitution of an Ala for Arg-77 in GapB resulted in a mutant protein with unique kinetic properties. While the activity of the mutant with NADH was comparable to any other GAPDH form (V_max about 50 µmol min^–1 mg^–1), the NADPH activity was lowered and completely insensitive to the redox poise (Fig. 2) in consequence of two independent effects: (1) the affinity for NADPH measured as K_m was 2.5-fold lower than in wild-type GapB (Table I); and (2) V_max(NADPH) under any redox condition (46 µmol min^–1 mg^–1) was similar to V_max(NADPH) of oxidized GapB (50 µmol min^–1 mg^–1) and in line with NADH-dependent reaction rates. The R77A mutation introduced in GapB thus appears to block the protein in a permanent unactivated state with catalytic properties similar to oxidized GapB.

The autoinhibitory function of the CTE of GapB has been proposed to be mediated by negative charges present in this mobile extension and an array of positive charges located on the S-loop (Fig. 1). In mutant B(E362Q), the negatively charged side chain of C-terminal Glu-362 was converted into a neutral side chain (Gln). The mutation had no effect on the affinity for substrate or coenzyme (Table I) but made B(E362Q) completely redox-insensitive. As a consequence, the NADPH activity remained at maximal levels under oxidizing conditions. In remarkable contrast to B(R77A), mutant B(E362Q) thus behaved as a constitutively "activated" form reproducing the kinetic properties of reduced GapB (Fig. 2).

Redox insensitivity of B(E362Q) was not due to the absence of the regulatory disulfide in the oxidized protein. Oxidized B(E362Q) was in fact less reactive with the thiol-labeling reagent monobromobimane by a factor of 1.7 ± 0.1 in respect to the reduced protein, indicating that the mutation did not hamper the formation of the regulatory disulfide.

Quaternary Structures

It is well known that the aggregation state of native AB-GAPDH depends on the type of pyridine nucleotide bound to the protein (Pupillo and Giuliani Piccari, 1975). In the presence of NADP, the tetrameric conformation A₂B₂ is stabilized (apparent molecular mass 189 kD in size exclusion chromatography; Fig. 3), while in the presence of NAD, the enzyme aggregates to an oligomer with 4-fold higher molecular mass (760 kD), strongly suggesting A₈B₈ stoichiometry. The CTE of GapB is required for NAD-mediated aggregation (Baalmann et al., 1996; Li and Anderson, 1997) and, in fact, B(minCTE) (151 kD) as well as GapA (132 kD) failed to aggregate in the presence of NAD and eluted as tetramers in the presence of either NAD or NADP (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Figure 3. Molecular mass estimates of eight GAPDH forms by size exclusion chromatography. Separations were performed on a Superdex 200 column (HR 10/30) equilibrated with either 0.2 mM NADP (white bars) or 0.2 mM NAD (black bars). Protein samples of 0.2 mL were loaded for every run. For every sample, molecular mass estimations were based on the elution volume of the absorbance peak at 280 nm. Mean values of duplicate experiments, with error bars representing SDs.

In the presence of NAD, mutant A(plusCTE) generated an unexpectedly high molecular mass form (>1.8 MD), at least 7-fold bigger than the tetramer; Fig. 3). Under the same conditions, all GapB forms (wild type and mutants) associated to oligomers ranging from 491 (wild type) to 553 kD [B(S188A)], compatible with octameric structures.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
NADP-dependent photosynthetic GAPDHs are present in every organism featuring oxygenic photosynthesis. Such archaic photosynthetic groups as cyanobacteria, red algae, and green microalgae contain a simple tetrameric NADP-GAPDH only constituted by A-type subunits (Figge et al., 1999). In some of these organisms including Synechocystis PCC6803 (Wedel and Soll, 1998), Chlamydomonas reinhardtii (Graciet et al., 2003), and Scenedesmus obliquus (Nicholson et al., 1987), the regulation of GAPDH appears to involve the reversible formation of a supramolecular complex between GAPDH and phosphoribulokinase (PRK), mediated by the small photosynthetic protein CP12.

In higher plants and certain green algae (e.g. Coleochaete and Chara), a second subunit of photosynthetic GAPDH (GapB) came on the scene as a likely product of gene fusion between subunit-A and the C-terminal end of CP12 (Pohlmeyer et al., 1996). A pair of Cys in the CTE of GapB reversibly form an intrachain disulfide under the control of thioredoxins. Disulfide-bearing CTE acts as an autoinhibitory domain that gives rise to a further, autonomous mechanism of GAPDH regulation (i.e. CP12-independent) typical of GapB-containing isoforms, including native A₂B₂ and recombinant GapB (Baalmann et al., 1996; Li and Anderson, 1997; Sparla et al., 2002). In all plant species investigated, only the NADPH-dependent activity of GAPDH is regulated (Steiger et al., 1971; Scagliarini et al., 1993). The regulatory mechanism involves thioredoxin and a number of metabolic effectors, pyridine nucleotides and 1,3-bisphosphoglycerate in particular, which affect the reversible aggregation of A₂B₂ tetramers into partially inactive A₈B₈ oligomers (Pupillo and Giuliani Piccari, 1975; Trost et al., 1993; Baalmann et al., 1996). Similar to thioredoxin regulation, the change of quaternary structure is also a CTE-dependent event (Baalmann et al., 1996; Li and Anderson, 1997; Sparla et al., 2002). The autonomous regulation of AB-GAPDH in chloroplasts coexists with the regulatory supramolecular complex containing GAPDH, phosphoribulokinase, and CP12 (Scheibe et al., 2002). The diverging features of these two regulatory systems suggest distinct physiological roles connected to the light/dark status of the plant.

Here, we show that the redox sensitive, C-terminal extension of GapB is a versatile regulatory module, which can induce novel regulatory functions when transferred to GapA subunits. The chimeric mutant A(plusCTE) was found to be redox-sensitive and prone to aggregate in the presence of NAD, both typical responses of regulatory GAPDH isoforms (Sparla et al., 2002), but with certain peculiarities. Indeed, the NADH-activity of A(plusCTE) mutant was redox-sensitive rather than constitutive as in GapB. Moreover, a comparison between oligomers of GapB, AB-GAPDH, and A(plusCTE) formed in the presence of NAD (478, 759, and >1,800 kD, respectively; Fig. 3) suggests that A(plusCTE) produced aggregates of molecular size at least double that of AB-GAPDH and comprised of 30 subunits or more. Although the B(minCTE) sequence is 80% identical to GapA, clearly B-subunits do not behave like A-subunits fused with CTE, and, judging from the relatively small size of GapB aggregates (478 kD, B₈), the CTE-dependent change in quaternary structure is enhanced by the presence of A-subunits.

GapA of spinach chloroplasts is the only type of photosynthetic GAPDH whose tridimensional structure has been solved to date (Fermani et al., 2001; Falini et al., 2003; Sparla et al., 2004) and provides the best approximation of the structure of GapB tetramers (CTE excluded). In GapA tetramers, A-subunits are symmetrically distributed with respect to three perpendicular axes (P,Q, and R; Fig. 4). Each subunit is constituted by a coenzyme binding domain and a catalytic domain. Coenzyme molecules, either NADP or NAD, bind to the coenzyme site of each subunit and protrude the catalytically active nicotinamide toward the active site (Fig. 4).

View larger version (89K): [in this window] [in a new window]   Figure 4. Ribbon model of the tridimensional structure of recombinant GapA from spinach (Sparla et al., 2004). Overview of the tetramer along the molecular P axis. The top-left subunit is colored dark gray; the other three are light gray. Four bound NADP molecules are schematically represented. For the sake of clarity, in one subunit only (top-right subunit, colored light gray) NADP is colored black and the side chains of Arg-77 (belonging to the same subunit) and Ser-188 (belonging to the R-axis related subunit, colored dark gray) are represented in sticks and balls. These two residues interact with the 2'-phosphate group of NADP. Identical interactions occur in all other subunits (not shown). The image was produced using MOLSCRIPT (Kraulis, 1991).

In mutant B(R77A), the redox regulation is completely abolished and NADPH activity is decreased to the level of NADH activity (about 50 µmol min^–1 mg^–1). The role of Arg-77 thus seems to be crucial in both coenzyme recognition and redox regulation. Mutant B(E362Q), too, is redox-insensitive, but the terminal residue Glu-362 is probably not involved in coenzyme discrimination since its NADPH-activity is higher than the NADH-dependent one, as usual. Therefore, differences between mutant B(E362Q) and wild-type GapB become apparent only under oxidizing conditions, which selectively depress the NADPH-activity of GapB without affecting the activity of mutant B(E362Q). Mutants B(R77A) and B(E362Q) mimic two opposite redox states of regulatory GapB: B(R77A) is permanently inhibited and reminiscent of oxidized GapB, B(E362Q) is fully "activated" (i.e. deinhibited) like reduced GapB. Mutant B(S188A) is intermediate in behavior, an intriguing parallel to the redox responses of A(plusCTE) construct (Fig. 2).

To explain all these results, we propose a model in which the elevated NADPH-dependent GAPDH catalysis depends on an optimal conformation adopted by the enzyme when NADPH is properly bound and held in place in the coenzyme site. In any other case, catalytically less effective conformations will be reached, as occurs during NAD(H)-dependent reactions and following CTE disulfide formation in thioredoxin-regulated GAPDH (with an approximately 2-fold drop in k_cat of NADPH-dependent reaction; Table I).

That an optimal enzyme conformation is associated with maximal NADPH-activity has been known since the structure of A₄-GAPDH complexed with NADP was solved at 2.0 Å resolution (Sparla et al., 2004). As an example of a less efficient conformation, on the other hand, mutant S188A of GapA exhibits a decreased NADPH-dependent activity and differs from wild-type GapA in that the tetramer is expanded by about 2 Å in each direction (8% volume increase). The fine structure of this mutant complexed with NADP revealed that the R77-2'phosphate interaction was lacking, so the S-loops did not reciprocally link the pairs of R-axis related subunits resulting in a relaxed conformation of the tetramer (Sparla et al., 2004). Why relaxed-versus-compact conformations should correspond to low-versus-high NADPH activities is far from being clear, but structural and functional data concur to support our model of GAPDH redox regulation as represented in Figure 5. In this scheme, full NADPH-activity of GAPDH is carried on by a compact tetramer ("fast" conformation) in which the bound NADPH is primarily sensed via interactions between the 2'-phosphate of the coenzyme and the side chain of Arg-77. GAPDH forms containing B-subunits with intact CTE have a fast conformation only under reducing conditions. Wild-type A₄-GAPDH and mutants B(E362Q) (Fig. 5) and B(minCTE) (as well as CTE-cys mutants of GapB; Sparla et al., 2002) do so under any redox condition and are permanently activated. In the oxidized CTE, on the contrary, the formation of a disulfide bridge forces the C-terminal Glu-362 to approach Arg-77, thereby distracting it from interactions with the 2'-phosphate of NADP. Under these conditions, Ser-188 and the opposite S-loop would quit their interacting position with the 2'-phosphate and the tetramer would relax to an expanded ("slow") conformation that catalyzes a slower reaction. This kinetic behavior is found not only in all oxidized GapB forms (except B(E362Q) and CTE-cys mutants), but also in mutants B(R77A) (this paper) and A(S188A) (Sparla et al., 2004) under any redox conditions. According to the model, the NADH-activity of GAPDH is not regulated because Arg-77 and Ser-188 do not interact with the coenzyme (Falini et al., 2003). However, in contrast to mutant A(S188A) complexed with NADP (Sparla et al., 2004), the conformation of NAD-complexed A₄-GAPDH is not "expanded" (Falini et al., 2003), although NADPH-dependent activities of inhibited forms are similar to NADH-dependent ones. Other structural constraints must explain the low efficiency of the NADH-activity. While tridimensional structures of oxidized GapB forms are not yet available, this work provides evidence that all GAPDH forms defective in coenzyme recognition do also share a kinetically inefficient conformation specifically affecting the NADPH-activity and possibly corresponding to the expanded tetrameric conformation of mutant A(S188A).

View larger version (22K): [in this window] [in a new window]   Figure 5. Conceptual scheme of redox regulation of GapB subunits based on perturbed redox regulation of site specific mutants. For the sake of clarity, each case is represented by a single GapB subunit. The change of location of the positively charged residue R77 (which interacts with either the negative charges of NADP 2'-phosphate or of C-terminal residue E362) determines a conformational change (slow or relaxed, fast or compact conformations). In this model, when R77 interacts with NADP (as in reduced GapB complexed with NADP or in B(E362Q) mutant under any redox condition) the conformation of the enzyme is compact (fast) and the catalytic efficiency is maximal. On the contrary, when R77 does not interact with NADP (in NADP-binding GapB under oxidizing conditions, and in mutant B(R77A) under any redox conditions), the enzyme conformation is relaxed (slow) and the catalytic efficiency is low. Residue S188, which interacts with NADP in concert with R77, is not shown for the sake of clarity. In the presence of NAD, there is no interaction between R77 and the coenzyme, and GapB displays low activity under any redox conditions, but the conformation of the tetramer might not be identical to the slow conformation of inhibited GapB forms. Therefore, the slow conformation of NAD-binding GAPDH is differently represented.

In land plants, dark inactivation of different GAPDH isozymes (A₄, A₂B₂) is achieved by different mechanisms with a common structural basis. Regulation of A₂B₂-isozyme depends on the CTE of subunit-B, which works as a sensor of the thioredoxin redox state in the stroma. The NADPH activity of A₂B₂-GAPDH is inhibited by oxidized thioredoxins and by NAD, the latter promoting the formation of A₈B₈ aggregates (Pupillo and Giuliani Piccari, 1975). In photosynthetic tissues, oxidized thioredoxins are more abundant in darkness than in the light (Buchanan and Balmer, 2005). Under the same conditions, NAD increases at the expenses of NADP (Muto et al., 1980; Heineke et al., 1991), an effect that might be ascribed to dark inactivation of NAD-kinase (Delumeau et al., 2000). The cooperation between oxidized thioredoxins and NAD in inactivating GAPDH is demonstrated by the fact that mutants with no functional sensor for thioredoxins [B(minCTE), this work; CTE-Cys mutants of GapB; Sparla et al., 2002] as well as native isozyme A₄ (Scagliarini et al., 1998) are all insensitive to NAD-induced inhibition/aggregation. Therefore, A₄-GAPDH, which may account for up to 30% of the total GAPDH activity in chloroplasts of higher plants (Cerff, 1979), requires another mechanism for dark inactivation. This mechanism is more ancient in evolution than CTE-based GAPDH regulation and involves CP12 and PRK as partner proteins (Wedel and Soll, 1998). Formation of the GAPDH/CP12/PRK complex in chloroplasts results in inhibition of enzyme activities and is favored in darkness (Scheibe et al., 2002). Oxidizing conditions and NAD promote complex formation in vitro (Graciet et al., 2004). The CTE of GapB derives in evolution from CP12 (Pohlmeyer et al., 1996) and both peptides share a similar C-terminal sequence, including the two redox-sensitive Cys (Sparla et al., 2002) and a C-terminal negatively charged amino acid (Glu-362 in spinach GapB; Asp-74 in spinach CP12; Pohlmeyer et al., 1996). The C-terminal portion of CP12 interacts with GAPDH (Wedel and Soll, 1998), and we propose that CP12 and CTE interact with GAPDH according to the same basic rules. Under oxidizing conditions, both CP12 and CTE have a conformation dictated by the presence of an internal disulfide bridge. This conformation forces a C-terminal negative charge (Glu-362 in CTE, Asp-74 in CP12) to approach Arg-77, thus distracting this residue from proper recognition of bound NADP. The consequence is enzyme inhibition, i.e. dark inactivation in vivo of any GAPDH isozyme.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Expression Vectors for Wild-Type and Mutant GAPDH Subunits

The cDNA fragments coding for subunits GapA, GapB, deletion GapB mutant lacking the C-terminal extension [B(minCTE); Baalmann et al., 1996], and chimeric GapA mutant bearing the CTE at the C terminus [A(plusCTE)] were transferred from pET-14 to pET-29 (Novagen) expression vectors. In short, the inserts were excised from pET-14 with BamHI and NdeI, purified from a 0.8% low melting agarose, and cloned into the BamHI/NdeI sites of the expression vector pET-29. The correct DNA sequence of all inserts cloned in pET-29 was controlled (Fig. 1).

Site-specific mutants B-E362Q, B-R77A, and B-S188A were constructed by PCR. The following primers, with mutations indicated in bold, were used according to the procedures of the QuickChange Site-Directed Mutagenesis kit (Stratagene): E362Q-L, 5'-GAGGAGTGCAAACTTTACCAGTAAGGATCCG-3'; E362Q-R, 5'-CGGATCCTTACTGGTAAAGTTTGCACTCCTC-3'; R77A-L, 5'-GGTTGTCTCTAACGCGGACCCTCTTAAACTTCCTTGGG-3'; R77A-R, 5'-CCCAAGGAAGTTTAAGAGGGTCCGCGTTAGAGACAACC-3'; S188A-L, 5'-CAGAGGCTGTGGGATGCTGCTCACAGGGACTTG-3'; S188A-R, 5'-CAAGTCCCTGTGAGCAGCATCCAACAGCCTCTG-3'.

The 50-µL PCR reactions contained 125 ng of each primer and 50 ng of template (pET29-GapB). Before transferring the plasmids into BL21(DE3) cells, the coding sequence of each mutant was sequenced.

Heterologous Expression and Protein Purification

Escherichia coli cells, strain BL21(DE3), were transformed with the above described constructs. Each E. coli BL21(DE3) strain harboring an expression plasmid was cultured in 20 mL Luria-Bertani medium supplied with kanamycin (50 µg/mL) at 37°C under vigorous shaking (130–150 rpm) for 16 to 18 h. Overnight cultures were transferred into 500 mL of fresh Luria-Bertani medium in the presence of kanamycin and grown under shaking at 37°C until an optical density of 0.4 to 0.8 at 600 nm was reached. The expression of recombinant proteins was induced by 0.4 mM isopropyl--D-thiogalactopyranoside. Three hours after induction, cells were harvested by centrifugation (10,000 g, 15 min). The pellet was washed with 25 mM K-phosphate, pH 7.5, 1 mM EDTA, 10 mM -mercaptoethanol, 1% (v/v) glycerol (buffer A), and spun down again before storage at –70°C for no longer than 2 d before use.

Cells were broken by lysozyme as described in Sparla et al. (2002). Shortly, the pellet was resuspended in buffer A supplemented with 0.5 mM NADP and 10 µg mL^–1 lysozyme. After 15 min incubation at room temperature, 1 µg mL^–1 DNase, 1 µg mL^–1 RNase, and 10 mM MgCl₂ were added to the sample, and incubation was prolonged for 20 min at 30°C. Cell debris was removed by centrifugation (20000g, 20 min) and the clear supernatant recovered for purification of recombinant proteins.

All recombinant proteins were purified by the same procedure. The supernatant obtained after cell lysis was first loaded on a Q-Sepharose HP 16/10 anion-exchange column (Pharmacia) equilibrated with buffer A. After thorough washing, bound proteins were eluted by 50 mL of linear K-phosphate gradient. The composition of the gradient was 25 to 500 mM for GapA and mutant B(minCTE), and 25 to 700 mM K-phosphate for any enzyme form containing the CTE that provides additional negative charges [GapB, A(plusCTE), B(S188A), B(R77A), and B(E362Q)]. Fractions containing NADPH-dependent GAPDH activity were pooled and desalted in buffer A. Samples were then loaded on a second anion-exchange column (MonoQ HR 5/5; Pharmacia) equilibrated with buffer A. Proteins were eluted by 15 mL of linear salt gradient (25–500 mM K-phosphate for GapA and B-CTE, and 25–700 mM K-phosphate for CTE-containing proteins). Fractions of 250 µL were collected and assayed for GAPDH activity. Active fractions were pooled, stored at +4°C, and used within the next 48 to 72 h.

Native AB-GAPDH was purified from spinach chloroplasts as previously described (Sparla et al., 2002).

Protein concentration in purified protein samples was determined by the Bradford method (Bradford, 1976) using bovine serum albumine as a standard.

Redox Titrations

For redox titration experiments, each enzyme form was desalted in 100 mM Tricine-NaOH, pH 7.9 (Sparla et al., 2002). Enzyme samples (about 2 pmol subunits) were incubated for 3 h at 25°C in the presence of an equimolar amount of recombinant E. coli thioredoxin (Sigma, St. Louis) and 20 mM dithiothreitol in different dithiol/disulfide ratios as described in Hutchinson and Ort (1995). Standard NAD(P)H-GAPDH activity was assayed after incubation in 50 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl₂, 3 mM 3-phosphoglycerate, 2 mM ATP, 5 U mL^–1 phosphoglycerate kinase (from rabbit muscle), and 0.2 mM NADPH. Oxidation of NADPH was followed at 340 nm with an ₃₄₀ of 6.23 mM^–1 (Falini et al., 2003). Activity data were fit by nonlinear regression (CoStat, CoHort Software) to the Nernst equation with an n value set at 2 (Hirasawa et al., 2000) and analyzed as described in Sparla et al. (2002). Midpoint redox potentials (E_m,7.9) are reported as mean values ± SD of at least three independent redox titrations performed under identical conditions and individually analyzed.

Disulfide formation in redox-insensitive mutant B(E362Q) was checked by labeling the enzyme with the fluorescent thiol-reagent monobromobimane (Hutchinson and Ort., 1995) following the modified procedure of Hirasawa et al. (1999).

Steady-State Kinetic Analysis

Steady-state kinetic analysis was accomplished by varying the concentration of NADPH between 250 and 5 µM (NADH between 300 and 10 µM) and 3-phosphoglycerate between 4 and 0.05 mM. The equilibrium concentration of the 1,3-bisphosphoglycerate was calculated on the basis of phosphoglycerate kinase K_eq of 3.1 x 10^–4 (Bergmeyer, 1985). Each kinetic experiment typically involved 70 single measurements of initial enzyme activity. Data were analyzed by nonlinear regression (CoStat) using a Michaelis Menten equation:

where S is the concentration of varying substrate and V_max(app) and K_m(app) are apparent kinetic constants. To obtain the limiting kinetic constants, the V_max(app) constants were interpolated by analogous nonlinear regression.

Kinetic parameters are given as mean±SD of at least three kinetic experiments performed under identical conditions.

Size Exclusion Chromatography

The molecular mass of purified recombinant proteins was estimated by size exclusion chromatography on a Superdex 200 HR 10/30 column as described in Sparla et al. (2002). Briefly, samples were desalted in 100 mM Tricine-NaOH, pH 7.9, and incubated on ice for 2 h in the presence of either 0.2 mM NADP (nonaggregating condition) or 0.2 mM NAD (aggregating condition). The column was equilibrated in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM KCl, 14 mM -mercaptoethanol, and 0.2 mM NADP or NAD. Elution was performed in the same buffer and optical absorbance was monitored at 280 nm. Fractions of 350 µL were collected and assayed for NAD(P)H-GAPDH activity. Apparent molecular mass was calculated on the basis of the elution volume of each 280-nm absorbance peak and the calibration of the column with ferritin (440 kD), catalase (232 kD), aldolase (158 kD), ovalbumin (43 kD), chymotrypsinogen A (25 kD), and ribonuclease A (13.7 kD) as standards (Pharmacia).

	ACKNOWLEDGMENTS

 
We thank Simona Fermani, Giuseppe Falini, and Alberto Ripamonti for helpful discussions.

Received March 2, 2005; returned for revision April 22, 2005; accepted April 22, 2005.

	FOOTNOTES

 
¹ This work was supported by Ministero dell'Istruzione, dell'Università e della Ricerca (PRIN 2003).

² These authors contributed equally to the paper.

³ Present address: Julius-Leber-Str. 27, 24145 Kiel, Germany.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.062117.

^* Corresponding author; e-mail trost{at}alma.unibo.it; fax 39–051–242576.

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