Characterisation of a phosphoenolpyruvate carboxylase kinase gene from Sorghum

Abstract

Light-induced phosphorylation of phosphoenolpyruvate carboxylase plays an important role in C4 photosynthesis. It reduces the sensitivity of phosphoenolpyruvate carboxylase to malate and thereby allows this enzyme to fix bicarbonate even in the presence of a high concentration of malate in the mesophyll cell cytosol. Previous work has shown that light causes an increase in the activity of phosphoenolpyruvate carboxylase kinase in a process involving protein synthesis. Phosphoenolpyruvate carboxylase kinase genes have been cloned from a range of C3 and CAM species, but not so far from C4 species. We have identified a phosphoenolpyruvate carboxylase cDNA from the C4 plant Sorghum bicolor. It encodes a 307 residue protein of predicted Mr 32524, which, like other phosphoenolpyruvate carboxylase kinases, comprises a protein kinase catalytic domain with minimal extensions at the N- and C-terminal ends. However, relative to other phosphoenolpyruvate carboxylase kinases, the Sorghum enzyme contains an acidic 23-residue insert just after the catalytic loop. Structure prediction suggests that this insert would be exposed at the surface of the protein. Using in vitro transcription and translation, we have demonstrated that the protein encoded by this cDNA has high phosphoenolpyruvate carboxylase kinase activity. This appears to be the first report of a cloned phosphoenolpyruvate carboxylase kinase from a C4 plant. Data on the expression of the gene in response to light and to cycloheximide will be presented.