Bacterial-type Phosphoenolpyruvate Carboxylase (PEPC) Functions as a Catalytic and Regulatory Subunit of the Novel Class-2 PEPC Complex of Vascular Plants

Abstract

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. Phosphoenolpyruvate (PEP)2 carboxylase (PEPC; EC 4.1.1.31) is an important enzyme of plant C metabolism that catalyzes the irreversible ␤-carboxylation of phosphoenolpyruvate to yield oxaloacetate and P i . This enzyme has been intensively studied with regards to its crucial role in catalyzing atmospheric CO 2 fixation in C 4 and crassulacean acid metabolism photosynthesis (1, 2). It also plays essential functions in bacteria and non-green plant cells, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates withdrawn for biosynthesis and N-assimilation (3, 4). Most vascular plant PEPCs exist as a homotetrameric "dimer of dimer" structure composed of four identical 100 -110-kDa subunits known as Class-1 PEPC. Class-1 PEPCs are subject to tight control by a combination of allosteric effectors and reversible phosphorylation at a conserved N-terminal seryl residue catalyzed by a dedicated Ca 2ϩ -independent PEPC protein kinase and protein phosphatase type 2A (1-3). Allosteric activation by hexose phosphates and inhibition by malate have been routinely observed, whereas phosphorylation activates the enzyme by reducing its sensitivity to malate inhibition and simultaneously enhancing activation by hexose phosphates (1-3). Our understanding of the post-translational control...

Three Different and Tissue-specific NAD-Malic Enzymes Generated by Alternative Subunit Association in Arabidopsis thaliana

“…In plants, multisubunit composition and changes in oligomeric assembly, depending on the tissue and the metabolic situation, are properties of important glycolytic and TCA cycle enzymes such as pyruvate kinase, NAD-isocitrate dehydrogenase, and PP i -dependent phosphofructokinase and phosphoenolpyruvate carboxylase (35)(36)(37)(38)(39). These characteristics represent a mechanism of allosteric regulation of enzymes of central metabolic pathways (40).…”

Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea

“…In contrast, BTPC genes encode larger 116-to 118-kD polypeptides owing to a unique intrinsically disordered region that mediates BTPC's tight interaction with coexpressed PTPC subunits. This association results in the formation of unusual Class-2 PEPC heterooctameric complexes that are largely desensitized to allosteric effectors and that dynamically associate with the surface of mitochondria in vivo (O'Leary et al, 2009(O'Leary et al, , 2011aIgawa et al, 2010;Park et al, 2012).…”

“…Class-1 PEPCs also fulfill a wide range of crucial nonphotosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis (O'Leary et al, 2011a). Class-1 PEPCs are subject to a complex set of posttranslational controls including allosteric effectors, covalent modification via phosphorylation or monoubiquitination, and protein-protein interactions (Uhrig et al, 2008;O'Leary et al, 2009O'Leary et al, , 2011aO'Leary et al, , 2011b.…”

Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation

“…Whereas most PEPCs exist as homotetramers (class 1 PEPC) (1), bacterial-type PEPCs associate with plant-type isozymes to form a heteromeric complex (class 2 PEPC) in unicellular green algae (11) and castor oilseeds (12). It has been recently shown that Class-2 PEPC is composed of four each of the bacterial-and plant-type isozymes (13).…”