In vivo regulatory phosphorylation of bacterial-type phosphoenolpyruvate carboxylase from developing castor oil seeds
O'LEARY, BRENDAN MICHAEL
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PEPC [PEP(phosphoenolpyruvate) carboxylase] is an essential and tightly controlled enzyme located at the core of plant C-metabolism. It fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and N-assimilation. In plants, a small multigene family encodes several closely related plant-type PEPC (PTPC) isozymes along with a distantly related bacterial-type PEPC (BTPC) isozyme. The PTPCs are well studied ~110-kDa subunits that typically exist as a homotetramer (Class-1 PEPC). By contrast, little is known about the larger ~118-kDa BTPC isozyme except that it occurs in developing castor (Ricinus communis) endosperm in tight association with PTPC subunits as a ~900-kDa hetero-octameric complex (Class-2 PEPC) that is greatly desensitized to metabolic effectors compared to Class-1 PEPC. This thesis elucidates the physiological purpose of the BTPC subunits by examining their structure/function relationship within Class-2 PEPC and identifying mechanisms of post-translational control. Recombinant expression and purification of the castor bean BTPC revealed unusual physical and kinetic properties including a remarkable insensitivity to metabolic effectors and a dependence upon PTPC subunits for structural stability. The first purification of a non-proteolyzed plant Class-2 PEPC complex was performed, and the kinetic analysis determined that the BTPC and PTPC subunits have complimentary catalytic properties. The BTPC subunits’ high Km(PEP) and desensitization to metabolic effectors may function as a metabolic overflow mechanism for sustaining flux from PEP to malate when PTPC subunits become feedback inhibited. An anti-PTPC co-immunopurification strategy was utilized to highly enrich non-proteolyzed BTPC from developing castor endosperm for downstream immunological and mass spectrometric analysis. BTPC was in vivo phosphorylated at multiple novel sites, identified by mass spectrometry as Thr4 or 5, Ser425 and Ser451. Phosphosite-specific antibodies towards Ser425 and Ser451 confirmed the existence of these sites in vivo and comparisons of Ser425 phosphorylation patterns established that the castor BTPC and PTPC phosphorytation sites are regulated independently. Phosphomimetic mutants of Ser425 caused BTPC inhibition by increasing its Km(PEP) and sensitivity to feedback inhibition. These results establish a novel mechanism of PEPC control whose implications within plant carbon metabolism are discussed.