Bacterial-type phosphoenolpyruvate carboxylase (PEPC) functions as a catalytic and regulatory subunit of the novel class-2 PEPC complex of vascular plants.
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 bacterial-type 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.
O'Leary
Brendan
B
Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Rao
Srinath K
SK
Kim
Julia
J
Plaxton
William C
WC
D017360:Arabidopsis / Q000378:metabolism; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000378:metabolism; D056890:Eukaryota / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008954:Models, Biological; D009584:Nitrogen / Q000737:chemistry; D010730:Phosphoenolpyruvate Carboxylase / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
D011994:Recombinant Proteins; D002244:Carbon; D010730:Phosphoenolpyruvate Carboxylase; D009584:Nitrogen