The isozymes of photosynthetic phosphoenolpyruvate carboxylase from C₄ plants (PEPC-C₄) play a critical role in their atmospheric CO₂ assimilation and productivity. They are allosterically activated by phosphorylated trioses or hexoses, such as d-glucose 6-phosphate, and inhibited by l-malate or l-aspartate. Additionally, PEPC-C₄ isozymes from grasses are activated by glycine, serine, or alanine, but the allosteric site for these compounds remains unknown. Here, we report a new crystal structure of the isozyme from Zea mays (ZmPEPC-C₄) with glycine bound at the monomer-monomer interfaces of the two dimers of the tetramer, making interactions with residues of both monomers. This binding site is close to, but different from, the one proposed to bind glucose 6-phosphate. Docking experiments indicated that d/l-serine or d/l-alanine could also bind to this site, which does not exist in the PEPC-C₄ isozyme from the eudicot plant Flaveria, mainly because of a lysyl residue at the equivalent position of Ser-100 in ZmPEPC-C₄ Accordingly, the ZmPEPC-C₄ S100K mutant is not activated by glycine, serine, or alanine. Amino acid sequence alignments showed that PEPC-C₄ isozymes from the monocot family Poaceae have either serine or glycine at this position, whereas those from Cyperaceae and eudicot families have lysine. The size and charge of the residue equivalent to Ser-100 are not only crucial for the activation of PEPC-C₄ isozymes by neutral amino acids but also affect their affinity for the substrate phosphoenolpyruvate and their allosteric regulation by glucose 6-phosphate and malate, accounting for the reported kinetic differences between PEPC-C₄ isozymes from monocot and eudicot plants.

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