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- Title
Comparison of Plant-Type Phosphoenolpyruvate Carboxylases from Rice: Identification of Two Plant-Specific Regulatory Regions of the Allosteric Enzyme.
- Authors
Masayuki Muramatsu; Rintaro Suzuki; Toshimasa Yamazaki; Mitsue Miyao
- Abstract
Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae and vascular plants, and it undergoes allosteric regulation by various metabolic effectors. Rice (Oryza sativa) has five plant-type PEPCs, four cytosolic and one chloroplastic. We investigated their kinetic properties using recombinant proteins and found that, like most plant-type PEPCs, rice cytosolic isozymes were activated by glucose 6-phosphate and by alkaline pH. In contrast, no such activation was observed for the chloroplastic isozyme, Osppc4. In addition, Osppc4 showed low affinity for the substrate phosphoenolpyruvate (PEP) and very low sensitivities to allosteric inhibitors aspartate and glutamate. By comparing the isozyme amino acid sequences and three-dimensional structures simulated on the basis of the reported crystal structures, we identified two regions where Osppc4 has unique features that can be expected to affect its kinetic properties. One is the N-terminal extension; replacement of the extension of Osppc2a (cytosolic) with that from Osppc4 reduced the aspartate and glutamate sensitivities to about one-tenth of the wild-type values but left the PEP affinity unaffected. The other is the N-terminal loop, in which a conserved lysine at the N-terminal end is replaced with a glutamate-alanine pair in Osppc4. Replacement of the lysine of Osppc2a with glutamate- alanine lowered the PEP affinity to a quarter of the wildtype level (down to the Osppc4 level), without affecting inhibitor sensitivity. Both the N-terminal extension and the N-terminal loop are specific to plant-type PEPCs, suggesting that plant-type isozymes acquired these regions so that their activity could be regulated properly at the sites where they function.
- Subjects
CARBOXYLASES; ALLOSTERIC enzymes; RICE; VASCULAR plants; PLANT metabolism; ALLOSTERIC regulation; CHLOROPLASTS; PHYSIOLOGY
- Publication
Plant & Cell Physiology, 2015, Vol 56, Issue 3, p468
- ISSN
0032-0781
- Publication type
Article
- DOI
10.1093/pcp/pcu189