Heat Stability of Maize Endosperm ADP-Glucose Pyrophosphorylase Is Enhanced by Insertion of a Cysteine in the N Terminus of the Small Subunit

Identification of Motifs Important in Heat Stability

Sequence alignments of heat-stable and heat-labile AGPase small subunits revealed pronounced sequence conservation (Hannah et al., 2001). However, significant sequence variation occurs in both termini. Of particular interest was the N-terminal motif QTCL (Fig. 1A). This sequence motif occurs only in the heat-stable AGPases shown in Figure 1 and Hannah et al. (2001). The Cys residue in this motif has been implicated in conferring heat stability to the potato tuber AGPase (Ballicora et al., 1999). To establish whether this motif bestows heat stability in the otherwise heat-labile maize endosperm AGPase, four variants were created in the N terminus of the maize endosperm small subunit (Fig. 1B). The mutation STCL replaces the native endosperm Tyr residue with a Cys. The mutation termed QTCL adds a Gln residue and changes the Tyr residue to a Cys. To evaluate any alteration caused by the Gln insertion, two additional constructs were created, ETCL and QTYL. Each recombinant AGPase small subunit plus a wild-type maize endosperm large subunit on a separate, compatible vector was expressed in an E. coli mutant harboring an inactive AGPase. Functional AGPase encoded by the plasmids complements the mutant E. coli, resulting in glycogen production. This is easily detected by brown staining of colonies following exposure to iodine vapors. All variant small subunits were functional in E. coli (Fig. 2).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Alignment of small subunits and created mutations. A, Position of the first amino acid shown is given in parentheses following the small subunit origin. Sequence numbering is based on the maize endosperm small subunit. Maize and barley endosperm are heat sensitive while maize embryo, maize leaf, and potato tuber are heat stable. Amino acids examined here are in bold and boxed. Shaded areas indicated highly conserved amino acids. B, Changes in the N termini of the various mutants are listed. Inserted amino acids are in bold.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Staining of mutations. Cells were grown overnight and then exposed to iodine vapors for 1 min. Complementation is observed by the production of brown staining. The positive control (WT) is wild-type maize endosperm AGPase, and the negative control (NEG) is cells containing empty vectors.

In agreement with the in vivo-produced glycogen, AGPase activity levels of the variants were comparable to that of the recombinant maize enzyme (Table I). AGPase activity from whole cell extracts was measured in both the forward and backward direction. Activity levels of the variants were moderately increased compared to the recombinant wild-type enzyme.

To determine whether any of the mutations conferred heat stability, whole cell extracts were incubated for 6 min at 58°C, and the percentage of AGPase activity remaining was determined. All constructs containing the added Cys (ETCL, QTCL, and STCL) exhibited significant increases in heat stability. Cys-containing variants retained 30% to 50% of AGPase activity (Table II), compared to only 2% for those without the Cys (wild type and QTYL). In addition, the Gln within this motif may aid in enhancing heat stability of mosaic subunits containing the added Cys.

Kinetic Analysis

Because of its increased activity and heat stability, detailed kinetic analysis was performed on the QTCL variant. Recombinant QTCL and wild-type enzymes were purified as described previously (Boehlein et al., 2005). K_m and V_max values for ATP and for G-1-P were determined for both enzymes in the presence of 3-PGA (Table III, first two entries). An inspection of the K_m values shows that the QTCL alteration has little to no effect on these parameters, indicating that this mutation does not have a direct role in substrate binding or catalysis. The alteration does affect catalytic efficiency as evidenced by the doubling of V_max of the purified enzyme. An increased V_max without changes in K_m values was also noted in the absence of 3-PGA (data not shown).

Allosteric properties of QTCL were also examined. Since the maize endosperm AGPase is activated by 3-PGA and deactivated by Pi, the K_a value for 3-PGA and the K_i value for Pi were determined. Resulting data show that the QTCL motif causes a small, but significant, increase in 3-PGA sensitivity (Table IV). The extent of phosphate modulation was determined in the presence of 3-PGA. Comparable K_i values for QTCL and wild-type recombinant AGPase were detected.

QTCL Disulfide Bridge Formation

The Cys of QTCL has been shown to be involved in disulfide bridge formation between the two small subunits (Jin et al., 2005). Because the wild-type maize endosperm lacks this Cys and the QTCL variant contains it, both were monitored for disulfide bond formation. Purified recombinant wild-type and QTCL AGPases were subjected to a nondenaturing SDS-PAGE analysis, blotted, and probed with antibodies against the large or small AGPase subunit. Proteins recognized by these antibodies are approximately 50 and 100 kD, the sizes of monomers and dimers of the two subunits, respectively (Fig. 3). Exposure to dithiothreitol (DTT) prior to electrophoresis abolishes the 100-kD proteins and intensifies the 50-kD band. We conclude that disulfide bridges maintain dimers not only of the small subunit, but also of the large subunit. Heterotetrameric AGPase was not observed in the presence or absence of DTT on the SDS gels.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Disulfide bridge formation. A nonreducing 10% SDS-PAGE gel followed by a western transfer was performed to investigate the formation of the disulfide bridge. A, Western blot developed with polyclonal small subunit. B, Western blot probed with a polyclonal large subunit antibody. Both antibodies recognize a dimer in QTCL and wild-type AGPase that depolymerizes in the presence of DTT. The small subunit dimer of QTCL is substantially more intense than that of wild type.

The presence of QTCL in the maize small subunit does not alter the size or amounts of proteins recognized by the large subunit antibody (Fig. 3B); however, its presence dramatically enhances the amount of the small subunit in the 100-kD protein at the expense of the 50-kD protein. Because the dimer involving the small subunit is abolished by the reducing agent and is enriched by the addition of the Cys in the N terminus, a disulfide bridge involving the small subunit is involved in dimerization. The low-intensity 100-kD band detected with the small subunit polyclonal antibody in wild type was shown to be nonspecific binding. This band is absent when an identical blot is developed with a monoclonal small subunit antibody (data not shown). We suspect that the cross-reaction of the polyclonal small subunit antibody with the large subunit is due to sequence conservation between the subunits.

Incorporation of the QTCL motif into the small subunit promotes dimer formation between small subunits but not between small and large subunits. Perusal of the two blots in Figure 3 shows that QTCL enhances the amount of the dimer detected with the small subunit antibody but not the dimer detected with large subunit antibody. Were heterodimers enhanced by the QTCL mutation, an increased intensity of both dimers would have been seen. We also identified a disulfide bond involving the large subunit in our purified extracts.

Activation of QTCL by DTT in the Absence of 3-PGA

Reduction of the Cys residue in the QTCL motif may represent a form of physiological control of the potato tuber AGPase (Ballicora et al., 2000; Tiessen et al., 2002). In the absence of 3-PGA, potato tuber AGPase is activated by preincubation with DTT and the substrate ADP-Glc (Fu et al., 1998). Accordingly, DTT and substrate modulation of recombinant maize endosperm AGPase containing or lacking the QTCL small subunit motif was investigated. Activities of the two AGPases were measured after preincubation with 3 mm DTT and/or 2.0 mm ADP-Glc (Fig. 4A). Preincubation of the QTCL variant with DTT and/or ADP-Glc enhanced activity almost 3-fold; however, wild-type AGPase was only slightly affected. While the greatest enhancement of QTCL occurred when both DTT and ADP-Glc were present, the modulating effects of the two are not additive (Fig. 4A). Interestingly, activation by DTT and/or ADP-Glc is not obligatory for maximal activity since the presence of saturating concentrations of the activator 3-PGA during the reaction more than compensates for preincubation activation (Fig. 4B).

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Reduction of the disulfide bridge. Reactions were incubated for 15 min at room temperature in 20 μL of a mixture containing 100 mm HEPES, pH 7.4, 0.2 mg/mL BSA, 5 mm MgCl₂, and the specified treatment listed. Following preincubation, reactions were performed using assay B with the following modification. The ADP-Glc concentration was adjusted from 2.0 mm so that the final concentration in all assays was 1.4 mm. The concentration of DTT used was 3.0 mm. Each assay was performed from 0 to 6 min and the slope of the velocity versus time plot was used to calculate the specific activity. A, Activity (μmol min⁻¹ mg⁻¹) in the absence of 3-PGA. Black bars represent wild-type and cross-hatching QTCL. B, Activity (μmol min⁻¹ mg⁻¹) of QTCL in the absence (white bars) or presence (gray bars) of 10 mm 3-PGA.

QTCL Heterotetramer Formation

Addition of QTCL to the N terminus of the small subunit confers substantial heat stability to the recombinant maize endosperm AGPase and enhances homodimer formation, as shown above. Next, the aggregation state of purified AGPase was monitored at various stages of heat inactivation (Fig. 5). Purified recombinant AGPases were incubated at 42°C for varying times and the levels of activity and their aggregation states were determined. The half-life of AGPase activity was increased approximately 10-fold at this temperature by addition of the QTCL motif, from 1.50 min to 13.0 min (Fig. 5A). Blue native (BN)-PAGE gels were used to investigate the aggregation state of the enzyme as a function of inactivation (Fig. 5B). Both AGPases exist primarily in the heterotetrameric state before exposure to elevated temperature. However, associated with the rapid loss of activity in wild type is the production of monomers and dimers. The heterotetrameric band is virtually abolished by 20 min, and the only proteins detected after this time are high-M_r aggregates. In contrast, the QTCL enzyme remains predominantly as a heterotetramer, even after a 30-min heat treatment. Note that bovine serum albumin (BSA) was not added to these samples to stabilize the activity of the enzymes; therefore, the half-life of QTCL at 42°C appears to be less than when incubated at 58°C. When this experiment was repeated with BSA, the half-life for the wild-type enzyme was approximately 4.5 min and the half-life for the QTCL mutant could not be determined due to its long stability over the course of the 45-min experiment (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

Heat stability of purified QTCL at 42°C. Enzymes were placed in a water bath at 42°C for varying times and then cooled on ice. Each enzyme was assayed for 10 min in the forward direction in the presence of 10 mm 3-PGA. Reactions were started with 0.065 μg of enzyme. Data were plotted as log% activity versus time (min), and the inactivation constant t_1/2 was calculated as follows: slope = −k/(2.3). t_1/2 is calculated from the equation k = 0.693/t_1/2. A, Inactivation time course at 42°C. Symbols are as follows: ▪, QTCL mutant; and ▴, wild-type enzyme. B, BN-PAGE gel transferred and probed with small subunit antibody at the time points from A. T, The approximately 220-kD tetramer; D, the approximately 100-kD dimer; and M, the approximately 50-kD monomer.

Further Small Subunit Modifications

Boehlein et al. (2005) recently reported an AGPase small subunit mosaic that contains amino acids 1 to 199 of the maize small subunit and 200 to 475 from the potato small subunit (MPss). Compared to the recombinant wild-type maize endosperm AGPase, this mosaic was recalcitrant to phosphate inhibition and exhibited increased heat stability. The half-life was 18 min at 42°C or 4 times greater than wild type. To determine whether there was an additive effect in heat stability, the QTCL mutation was placed into MPss. This new variant, termed QTCL-MP, was kinetically indistinguishable from the QTCL mutation (Table III). Interestingly, the increased 3-PGA sensitivity and decreased Pi inhibition caused by substituting the terminal 275 potato tuber small subunit amino acids for their counterparts of maize were nullified by the N-terminal QTCL mutation (Table IV). The QTCL-MP mutation is approximately 5 times more heat stable than QTCL and over 300 times more stable than the recombinant wild-type maize endosperm enzyme (Fig. 6).

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

Heat stability of purified QTCL, wild type, and QTCL-MP at 55°C. Reactions were carried out as described in Figure 5, except temperature was raised to 55°C. ▪, Wild type; ⋄, MP; ▵, QTCL; ∇, QTCL-MP.

Site-Directed Mutagenesis

Mutations in the maize (Zea mays) small subunit were created essentially as described by Horton et al. (1993). PCR-mutagenized DNA fragments of the maize endosperm small subunit were digested with NcoI and KpnI. These were used to replace the equivalent wild-type region of small subunit in an expression vector. Mutations were verified by sequencing. The vector was transformed into the Escherichia coli strain AC70R1-504, which also contained the wild-type large subunit coding region on a compatible expression vector (Giroux et al., 1996). The AC70RI-504 cell line contains a mutation that renders the strain incapable of producing bacterial AGPase (Iglesias et al., 1993).

Growth and Purification of Maize AGPase from E. coli

Protein inductions were as described by Burger et al. (2003), except that induction was performed for 3 h at room temperature. Following cell harvest by centrifugation, cell pellets were stored at −80°C.

Enzyme Purification

Purification of the wild type and the QTCL variant AGPase was done as described by Boehlein et al. (2005). For crude extracts, the bacterial pellets were resuspended in 1.0 mL of extraction buffer (50 mm HEPES, pH 7.5, 200 mm KCl, 10 mm MgCl₂, 2.5 mm EDTA, and 5% Suc) containing 20% ammonium sulfate, 50 μg/mL lysozyme, 1 μg/mL pepstatin, 1 μg/mL leupeptin, 1 mm phenylmethylsulfonyl fluoride, 10 μg/mL chymostatin, and 1 mm benzamidine. The lysate was maintained on ice and sonicated three times for 10 s each. The sample was centrifuged for 5 min at 12,500 rpm at 4°C and the supernatant was conserved on ice. Solid ammonium sulfate was added to 45% saturation and the sample was centrifuged for 5 min at 12,500 rpm at 4°C. The pellet was resuspended in extraction buffer containing protease inhibitors and stored on ice. The concentration of the crude protein extract was determined using the Bio-Rad protein assay using BSA as a standard.

Assay A (Forward Direction, Radioactive)

For heat-stability measurements, the enzymes were diluted to 1.0 μg/μL and divided into two tubes. A single tube remained on ice while the second tube was placed at 58°C for 6 min with occasional gentle agitation. AGPase activity of the crude extracts was determined in the direction of ADP-Glc synthesis as described in Burger et al. (2003) with a reaction time of 5 min. Reactions were started by the addition of the enzyme. The value reported within an experiment is the average from triplicate samples.

Assay B (Reverse Direction, Nonradioactive)

A nonradioactive endpoint assay was used to measure G-1-P produced by coupling synthesis to NADH production using phosphoglucomutase and Glc-6-P dehydrogenase as described by Boehlein et al. (2005). The temperature of all the assays was 37°C unless otherwise specified. Standard reaction mixtures contained 100 mm MOPS HCl, pH 7.4, 0.4 mg/mL BSA, 5 mm MgCl₂, 1 mm ADP-Glc, 20 mm 3-PGA, 1 mm sodium pyrophosphate, and enzyme in 100-μL reaction volume. Reactions were incubated for 5 min and terminated by boiling in a water bath for 1 min. After termination, 330 μL of water were added, followed by 70 μL of a development mixture containing a final concentration of 100 mm MOPS HCl, pH 7.4, 0.1 mg/mL BSA, 7 mm MgCl₂, 0.6 mm NAD, 1 unit Glc-6-P dehydrogenase, and 1 unit phosphoglucomutase. Reactions were centrifuged for 5 min and then the absorbance read at 340 nm. Amount of G-1-P produced was calculated from a standard curve using freshly prepared G-1-P instead of enzyme. Assay tubes were prewarmed to 37°C prior to assaying. All assays were initiated by the addition of enzyme. Specific activity is defined as a unit/mg protein. Purification was always monitored using the reverse assay.

Assay C (Forward Reaction, Nonradioactive)

A nonradioactive endpoint assay was used to determine the amount of inorganic pyrophospate produced by coupling it to a decrease in NADH using a pyrophosphate reagent (Sigma P-7275) as previously described (Boehlein et al., 2005). Standard reaction mixtures contained 50 mm HEPES, pH 7.4, 15 mm MgCl₂, 4.0 mm ATP, and 4.0 mm G-1-P in 200 μL. 3-PGA was added at varying amounts, as specified. Reactions were terminated after 10 min by boiling in a water bath for 1 min. The reactions were developed by adding 300 μL of pyrophosphate reagent (one bottle diluted with 22.5 mL water) to each assay and then the absorbance read at 340 nm. Change in absorbance between the blank and the reaction was used to calculate the amount to inorganic pyrophospate produced. All reactions were linear with time and enzyme concentration. Assay tubes were prewarmed to 37°C prior to assay and were initiated by enzyme addition.

Enzyme Kinetics

Heat Stability

The half-life of wild type and QTCL at 42°C was determined by desalting enzyme in 50 mm HEPES, pH 6.5, 5.0 mm MgCl₂, 0.5 mm EDTA. Heat was applied to desalted enzyme (0.065 mg/mL) and, at the appropriate time, enzyme was withdrawn from the tube and placed on ice. This enzyme was used for activity assays and BN gels. All reactions were carried out in triplicate using assay C with the addition of 10 mm 3-PGA. Both SDS-PAGE and BN-PAGE gels were prepared as outlined (Boehlein et al., 2005). Briefly, the gradient for the BN-PAGE was 5% to 18%. The buffers used are according to Schagger et al. (1994) with minor modifications. Two types of cathode buffer were prepared, one contained 0.002% Coomassie G and the other lacked Coomassie G. Aminocaproic acid was not used in the gel buffer. The gels were run at 4°C for 20 min at 100 V in cathode buffer containing Coomassie G. The voltage then was increased to 200 V for an additional 20 min. Finally, the gel was transferred to cathode buffer without Coomassie and run at 200 V until the dye front was off the gel. The gel was equilibrated in cold 1× transfer buffer (25 mm Tris base, 192 mm Gly, and 20% methanol) plus 1% SDS. A standard western transfer procedure was performed using polyvinylidene difluoride membrane with a 0.2 μm pore size (Bio-Rad). The molecular mass markers were as follows: thyroglobulin, 669,000 kD; ferritin, 440,000 kD; catalase, 232,000 kD; lactate dehydrogenase, 140,000 kD; and BSA, 67,000 kD (Amersham-Pharmacia Biotech electrophoresis calibration kit). Each lane contained approximately 0.87 μg of purified enzyme. All BN-PAGE gel western blots were developed with polyclonal antibodies against the maize AGPase small subunit.

Heat stability experiments at 55°C were performed as described above, except each enzyme was desalted into 50 mm HEPES, pH 7.4, 5 mm MgCl₂, 0.5 mm EDTA followed by the addition of BSA (0.5 mg/mL). Data were plotted as log% activity versus time, and the inactivation constant t_1/2 was calculated as follows: slope = −k/(2.3). t_1/2 is calculated from the equation k = 0.693/t_1/2.

Nonreducing 10% SDS-PAGE gels were prepared according to standard procedures (Sambrook et al., 1989). Approximately 0.43 μg of purified enzyme was loaded in each lane, and western blots were developed using polyclonal antibodies raised against the small and large subunits of the maize AGPase.

Disulfide Bridge Reduction

Enzymes were desalted into 50 mm HEPES, pH 7.4, 5 mm MgCl₂, and 0.5 mm EDTA. Protein concentrations were determined and BSA was added to a final concentration of 0.5 mg/mL to stabilize the desalted enzyme. Enzymes were incubated for 15 min at room temperature in a mixture containing 100 mm HEPES, pH 7.4, 0.2 mg/mL BSA, 5 mm MgCl₂, and the appropriate treatment in a 20-μL volume. Following this incubation, reactions were performed using assay B with the following modification. The final concentration in samples with DTT was 3.0 mm and the ADP-Glc concentration was adjusted so that the final concentration in the assay was always 1.4 mm. Each assay was performed for 2, 3, 4, 5, and 6 min and the slope of the plot of velocity versus time was used to calculate the specific activity.