Research article
Two phosphoenolpyruvate carboxykinases coexist in the Crassulacean Acid Metabolism plant Ananas comosus. Isolation and characterization of the smaller 65 kDa form

https://doi.org/10.1016/j.plaphy.2011.02.015Get rights and content

Abstract

Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30 °C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.

Highlights

► Two PEPCK isoforms of 74 and 65 kDa were coexist in vivo in leaves of pineapple. ► Characterization of the 65-kDa PEPCK showed that it possess regulatory properties. ► The results suggest that it could participate in C and N metabolism as a decarboxylase.

Introduction

Phosphoenolpyruvate (PEP) carboxykinase (PEPCK) catalyses the ATP dependent (EC 4.1.1.49) or GTP dependent (EC 4.1.1.32) reversible conversion of OAA to PEP: OAA + A(G)TP  PEP + A(G)DP + CO2.

The forms of PEPCK that utilize only guanine (sometimes hypoxanthine) nucleotides and needs Mg2+ or Mn2+ for maximal activity are largely confined to vertebrates. On the other hand the enzymes present in higher plants, bacteria, yeast and trypanosomes utilize only, or preferentially, adenine nucleotides and have a strict requirement for Mn2+ [1]. Although they share considerable homology, the enzyme from higher plants is considerably larger than PEPCK from other organisms because it possesses an N-terminal extension that is rapidly lost by proteolysis upon preparation of crude extracts [1].

In plants, PEPCK has several important physiological functions: i) it catalyses a key step in the conversion of fats to sugars during the germination of fat-storing seeds [2], [3], [4]; ii) in a marine macroalga, the carboxylation reaction provides C4 acids to the chloroplast for decarboxylation, thus acting as a CO2 concentrating mechanism at the site of CO2 fixation via RuBisCO [5]; iii) it has been proposed to be involved in nitrogen and amino acid metabolism [6], [7] and; iv) acting as a decarboxylase, it provides CO2 to the reductive pentose phosphate pathway in PEPCK-type C4 and Crassulacean Acid Metabolism (CAM) plants [8], [9].

CAM metabolism involves the uptake of CO2 through the stomata at night and assimilation through PEP carboxylase (PEPC) into 4-carbon organic acids [10], [11]. During the day the C4 acids are decarboxylated to yield CO2, which is assimilated by RuBisCO and the reductive pentose phosphate pathway. In the leaves of CAM plants, in which PEPCK is the major decarboxylase, PEPC and PEPCK coexist in the cytosol of the same cells and the former needs to be active at night while the latter must be during the day. This fact suggests that to avoid a futile cycle of carboxylation/decarboxylation, there must be mechanisms that modulate both activities. PEPC's regulation has been thoroughly studied and understood [12], [13], but in comparison little is known about PEPCK regulation. One major hindrance to the comprehension of PEPCK’s regulatory properties is the extreme sensitivity of the enzyme to proteolysis during extraction from plant tissues, which leaves a shorter, yet active, form of the enzyme. Interestingly, while the truncated form of PEPCK shows sensitivity to some metabolites [14], [15], this is not enough to completely reveal its regulation in vivo. There is a high chance that the portion of the enzyme that is proteolytically cleaved off during extraction confers additional regulatory properties to the enzyme. In fact, native PEPCK is phosphorylated in some C4 leaves and in all CAM leaves and C3-tissues that have been studied to date [16], [17]. It is assumed that phosphorylation might occur at night, rendering the enzyme less active, although a connection between the phosphorylation status of PEPCK and a decreased activity in darkened leaves has only been demonstrated for the enzyme from the C4 plant Guinea grass [18], [19].

In higher plants, PEPCK has been purified to homogeneity from the leaves of C4 grasses [14], [18] and cucumber [20] and partially purified from the cotyledons of marrow [2] and from the leaves of the CAM bromeliad, pineapple (Ananas comosus L.) [21], [22]. In the latter case, the enzyme’s kinetic properties were partially characterized, although its molecular mass was not reported [21], [22].

In an attempt to further define PEPCK’s role in CAM photosynthesis, a 65 kDa PEPCK from pine apple leaves was purified to homogeneity and biochemically and physically characterized. A polyclonal antiserum against this truncated form has been raised and evidence is presented that strongly indicates that both 74-kDa and a 65-kDa forms of PEPCK exist in pineapple leaves in vivo.

Section snippets

PEPCK purification

The activity of PEPCK determined in the PEP-carboxylation direction in illuminated pineapple leaf crude extracts was 2.19 U g−1 fresh weight.

PEPCK was purified by a combination of column chromatography methods: tentacle anion exchange on Fractogel, affinity chromatography on HiTrap Chelating, and ion exchange on High Q. A single PEPCK activity peak was observed in the first two chromatographic procedures, although in the latter, PEPCK eluted in 3 peaks at 110, 160 and 190 mM KCl, that were named

Discussion

PEPCK is a ubiquitous enzyme involved in many aspects of plant metabolism. In certain CAM plants, in particular, it acts during the day as a decarboxylating enzyme that provides CO2 for the diurnal RuBisCO-dependent carbon fixation. Since plant PEPCK is cytosolic, as is PEPC, a reciprocal regulation of both activities is to be expected to avoid futile cycling of ATP and CO2. So far, the study of plant PEPCK properties has been hindered by the extreme proteolytic lability of the enzyme’s

Conclusion

Two phosphoenolpyruvate carboxykinase isoforms of 74 and 65 kDa were found to coexist in vivo in leaves of the CAM plant pineapple. Purification and characterization of the 65-kDa PEPCK showed that it is an active enzyme that displays some clear, albeit modest, regulatory properties. The results suggest that it could participate in carbon and perhaps nitrogen metabolism in vivo, acting as a decarboxylase. Its abundance relative to the larger 74-kDa form that is present in all C4 plants

Plant material

Pineapple fruits (Ananas comosus L. cv. Havaiano) were purchased at local markets. Detached crowns were placed in pots with tap water until they rooted. Plants under hydroponic culture were grown for 1 month in a greenhouse under a natural illumination regime of 16 h light/8 h dark, corresponding to the Southern hemisphere summer conditions. The temperature was 25–30 °C during the day and 17 °C during the night.

Fully developed pineapple leaves were harvested 6–8 h into the light period when the

Acknowledgments

FEP is a member of the Investigator Career and MM and SPR are Fellows of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). This work was supported by grants from Agencia Nacional de Promoción de Ciencia y Tecnología (PICT N°32459) and CONICET (PIP 2519).

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    Present address: Instituto de Biología Molecular y Celular de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina.

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