In Vitro Phosphorylation of Phosphoenolpyruvate Carboxylase from the Green Alga Selenastrum minutum (original) (raw)
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Biochemical Journal, 2011
PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO2 during C4 and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C4–C6 carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-...
Manipulating PEPC levels in plants
Journal of Experimental Botany, 2002
This review examines the current understanding of the structural, functional and regulatory properties of C4 and C3 forms of higher plant phosphoenolpyru- vate carboxylase. The emphasis is on the interactive metabolic and post-translational controls acting on the enzyme in the physiological context of C4 photo- synthesis and the anaplerotic pathway. A brief over- view is given concerning the recent developments
Biochemical and Biophysical Research Communications, 2001
In Crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) is subject to day-night regulatory phosphorylation of a conserved serine residue in the plant enzyme's N-terminal domain. The dark increase in PEPC-kinase (PEPC-k) activity is under control of a circadian oscillator, via the enhanced expression of the corresponding gene (1). The signaling cascade leading to PEPC-k upregulation was investigated in leaves and mesophyll cell protoplasts of the facultative, salt-inducible CAM species, Mesembryanthemum crystallinum. Mesophyll cell protoplasts had the same PEPC-k activity as leaves from which they were prepared (i.e., high at night, low during the day). However, unlike C 4 protoplasts (2), CAM protoplasts did not show marked PEPC-k up-regulation when isolated during the day and treated with a weak base such as NH 4 Cl. Investigations using various pharmacological reagents established the operation, in the darkened CAM leaf, of a PEPC-k cascade including the following components: a phosphoinositide-dependent phospholipase C (PI-PLC), inositol 1,4,5 P (IP 3)-gated tonoplast calcium channels, and a putative Ca 2؉ /calmodulin protein kinase. These results suggest that a similar signaling machinery is involved in both C 4 (2, 3) and CAM plants to regulate PEPC-k activity, the phosphorylation state of PEPC, and, thus, carbon flux through this enzyme during CAM photosynthesis.
PLANT PHYSIOLOGY, 1991
Control of C4 photosynthesis and Crassulacean acid metabolism (CAM) is, in part, mediated by the diel regulation of phosphoenolpyruvate carboxylase (PEPC) activity. The nature of this regulation of PEPC in the leaf cell cytoplasm of C4 and CAM plants is both metabolite-related and posttranslational. Specifically, the regulatory properties of the enzyme vary in accord with the physiological activity of C4 photosynthesis and CAM: PEPC is less sensitive to feedback inhibition by L-malate under light (C4 plants) or at night (CAM plants) than in darkness (C4) or during the day (CAM). While the view that a light-induced change in the aggregation state of the holoenzyme is a general mechanism for the diel regulation of PEPC activity in CAM plants is currentiy in dispute, there is no supportive in vivo evidence for such a tetramer/dimer interconversion in C4 plants. In contrast, a wealth of in vitro and in vivo data has accumulated in support of the view that the reversible phosphorylation of a specific, N-terminal regulatory serine residue in PEPC (e.g. Ser-15 or Ser-8 in the maize or sorghum enzymes, respectively) plays a key, if not cardinal, role in the posttranslational regulation of the carboxylase by light/ dark or day/night transitions in both C4 and CAM plants, respectively. Phosphoenolpyruvate carboxylase (PEPC2, EC 4.1.1.31) catalyzes the irreversible ,B-carboxylation of PEP in the pres
FEBS Letters, 2009
a b s t r a c t 26 Two phosphoenolpyruvate carboxylase (PEPC) kinase genes (PPCk1 and PPCk2) are present in the 27 Arabidopsis genome; only PPCk1 is expressed in rosette leaves. Homozygous lines of two independent 28 PPCk1 T-DNA-insertional mutants showed very little (dln1), or no (csi8) light-induced PEPC phos-29 phorylation and a clear retard in growth under our greenhouse conditions. A mass-spectrometry-30 based analysis revealed significant changes in metabolite profiles. However, the anaplerotic pathway 31 initiated by PEPC was only moderately altered. These data establish the PPCk1 gene product as 32 responsible for leaf PEPC phosphorylation in planta and show that the absence of PEPC phosphory-33 lation has pleiotropic consequences on plant metabolism.
Journal of Biological Chemistry, 2009
Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated anaplerotic enzyme situated at a major branch point of the plant C metabolism. Two distinct oligomeric classes of PEPC occur in the triglyceride-rich endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of identical 107-kDa plant-type PEPC (PTPC) subunits (encoded by RcPpc3), whereas the novel Class-2 PEPC 910-kDa hetero-octameric complex arises from a tight interaction between Class-1 PEPC and distantly related 118-kDa bacterialtype PEPC (BTPC) polypeptides (encoded by RcPpc4). Here, COS BTPC was expressed from full-length RcPpc4 cDNA in Escherichia coli as an active PEPC that exhibited unusual properties relative to PTPCs, including a tendency to form large aggregates, enhanced thermal stability, a high K m(PEP) , and insensitivity to metabolite effectors. A chimeric 900-kDa Class-2 PEPC hetero-octamer having a 1:1 stoichiometry of BTPC:PTPC subunits was isolated from a mixture of clarified extracts containing recombinant RcPPC4 and an Arabidopsis thaliana Class-1 PEPC (the PTPC, AtPPC3). The purified Class-2 PEPC exhibited biphasic PEP saturation kinetics with high and low affinity sites attributed to its AtPPC3 and RcPPC4 subunits, respectively. The RcPPC4 subunits: (i) catalyzed the majority of the Class-2 PEPC V max , particularly in the presence of the inhibitor L-malate, and (ii) also functioned as Class-2 PEPC regulatory subunits by modulating PEP binding and catalytic potential of its AtPPC3 subunits. BTPCs appear to associate with PTPCs to form stable Class-2 PEPC complexes in vivo that are hypothesized to maintain high flux from PEP under physiological conditions that would otherwise inhibit Class-1 PEPCs.
Archives of Biochemistry and Biophysics, 1989
A reconstituted system composed of purified phosphoenolpyruvate carboxylase (PEP-Case) and a soluble protein kinase (PK) from green maize leaves was developed to critically assess the effects of in vitro protein phosphorylation on the catalytic and regulatory (malate sensitivity) properties of the target enzyme. The PK was partially purified from light-adapted leaf tissue by ammonium sulfate fractionation (O-69% saturation fraction) of a crude extract and blue dextran-agarose affinity chromatography. The resulting preparation was free of PEPCase. This partially purified protein kinase activated PEP-Case from dark-adapted green maize leaves in an ATP-, Mgs'-, time-, and temperaturedependent fashion. Concomitant with these changes in PEPCase activity was a marked decrease in the target enzyme's sensitivity to feedback inhibition by L-malate. The PKmediated incorporation of "P from [y-32P]ATP into the protein substrate was directly correlated with these changes in PEPCase activity and malate sensitivity. The maximal molar 32P-incorporation value was about 0.25 per lOO-kDa PEPCase subunit (i.e., 1 per holoenzyme). Phosphoamino acid analysis of the 32P-labeled target enzyme by two-dimensional thin-layer electrophoresis revealed the exclusive presence of phosphoserine. These in vitro results, together with our recent studies on the light-induced changes in phosphorylation status of green maize leaf PEPCase in vivo (J.-A. Jiao and R. Chollet (1988) Arch. Biochem. Biophys. 261,409-41'7), collectively provide the first unequivocal evidence that the seryl-phosphorylation of the dark-form enzyme by a soluble protein kinase is responsible for the changes in catalytic activity and malate sensitivity of C, PEPCase observed in vivo during dark/light transitions of the parent leaf tissue. o 1989 Academic Press, Inc.
New Phytologist, 2007
www.newphytologist.org 775 Summary • Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C 4 -like photosynthesis system in the stems of this C 3 tree species. • The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (K m ) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. • Leaf and stem PEPCs exhibited similar, classical values characteristic of C 3 PEPCs, with an optimal pH of c. 7.8, a K m for PEP of c. 0.3 mM and a IC 50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the K m for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells.