Multiple highly expressed phosphoenolpyruvate carboxylase genes have divergent enzyme kinetic properties in two C4 grasses.

BACKGROUND AND AIMS: Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyses the irreversible carboxylation of PEP with bicarbonate to produce oxaloacetate. This reaction powers the carbon-concentrating mechanism (CCM) in plants that perform C4 photosynthesis. This CCM is generally driven by a single PEPC gene product that is highly expressed in the cytosol of mesophyll cells. We found two C4 grasses, Panicum miliaceum and Echinochloa colona, that each have two highly expressed PEPC genes. We characterized the kinetic properties of the two most abundant PEPCs in E. colona and P. miliaceum to better understand how the enzyme's amino acid structure influences its function. METHODS: Coding sequences of the two most abundant PEPC proteins in E. colona and P. miliaceum were synthesized by GenScript and were inserted into bacteria expression plasmids. Point mutations resulting in substitutions at conserved amino acid residues (e.g. N-terminal serine and residue 890) were created via site-directed PCR mutagenesis. The kinetic properties of semi-purified plant PEPCs from Escherichia coli were analysed using membrane-inlet mass spectrometry and a spectrophotometric enzyme-coupled reaction. KEY RESULTS: The two most abundant P. miliaceum PEPCs (PmPPC1 and PmPPC2) have similar sequence identities (>95 %), and as a result had similar kinetic properties. The two most abundant E. colona PEPCs (EcPPC1 and EcPPC2) had identities of ~78 % and had significantly different kinetic properties. The PmPPCs and EcPPCs had different responses to allosteric inhibitors and activators, and substitutions at the conserved N-terminal serine and residue 890 resulted in significantly altered responses to allosteric regulators. CONCLUSIONS: The two, significantly expressed C4Ppc genes in P. miliaceum were probably the result of genomes combining from two closely related C4Panicum species. We found natural variation in PEPC's sensitivity to allosteric inhibition that seems to bypass the conserved 890 residue, suggesting alternative evolutionary pathways for increased malate tolerance and other kinetic properties.

Kinetic variation in grass phosphoenolpyruvate carboxylases provides opportunity to enhance C4 photosynthetic efficiency.

The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C4 plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C4 plants is driven by PEPC's affinity for bicarbonate (KHCO3 ), which can be rate limiting when atmospheric CO2 availability is restricted due to low stomatal conductance. We hypothesize that natural variation in KHCO3 across C4 plants is driven by specific amino acid substitutions to impact rates of C4 photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured KHCO3 from 20 C4 grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in KHCO3 across these C4 grasses (24.3 ± 1.5 to 46.3 ± 2.4 µm), which significantly impacts modeled rates of C4 photosynthesis. Additionally, molecular engineering of a low-HCO3- affinity PEPC identified key domains that confer variation in KHCO3 . This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO3- affinity and C4 photosynthetic efficiency in important C4 crops.