Kinetic properties of recombinant phosphomimic mutant of Zea mays phosphoenolpyruvate carboxylase (ZmPEPCS15D)

Phosphoenolpyruvate carboxylase (PEPC) is the key enzyme for fixing atmospheric carbon dioxide in C4 and CAM plants, and in anaplerotic metabolism in most plants. It is regulated by reversible phosphorylation, and allosteric activation or inhibition. The regulatory role of phosphorylation at N-terminal serine residue (S15) is documented earlier. In this study PEPC was cloned from maize and engineered to generate phosphomimic mutant enzyme by conversion of the codon for Serine 15 to aspartate (PEPCS15D). In vitro study with recombinant wild-type PEPC and phosphomimic PEPCS15D showed that both the enzymes are active catalytically. Kinetics analysis showed that PEPCS15D enzyme has comparatively lower K-m value over WT PEPC, thus implying higher affinity towards its substrate PEP than the wild type PEPC. However, both WT PEPC and mutant PEPCS15D showed similar regulation with the allosteric activator (Glycine) and allosteric inhibitors (L-Aspartate or L-Malate). Thus, the engineered phosphomimic mutant PEPCS15D with high affinity for PEP will be useful to engineer rice and other C3 plants for enhancing photosynthesis and minimizing respiratory CO2 loss.