Phophoenolpyruvate carboxylase (PEPC) from a thermophilic caynobacterium, Synechococcus vulcanus

Abstract

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus and expressed in E.coli BL21 (DE3) under the control of T7 promoter. The purified fusion protein (recombinant SvPEPC) showed very unusual allosteric regulatory properties. The enzyme activity was strongly activated by Fruc-1,6-P2 and slightly stimulated by Gluc-6-P, Glu and Gln. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I0.5 for Asp was increased about 2-fold by Gluc-6-P while markedly decreased about 4-fold by Fruc-1,6-P2. These allosteric properties are not readily explainable by conventional allosteric models. It seems that Fruc-1,6-P2 and Gluc-6-P do not share the same binding site on SvPEPC protein. When SvPEPC was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated in planta transformatiom under the control of the cauliflower mosaic virus 35S promoter, growth hindrance occurred on the transformants plants grown on agar plate with 1% sucrose under white light. In the early stage of growth, about 10% of plants showed bleaching in leaves or cotyledons in a gradient manner. A part of them could grow on soil but decreased apical dominance was observed in the adult plants. Leaf or cotyledon bleaching may be resulted from the exhaust of PEP due to the over-expression of SvPEPC in Arabidopsis thaliana.