Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thalianaα2β2-heterotetrameric pyruvate dehydrogenase (E1) plus A. thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in kcat, Km, and kcat/Km values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α

Molecular cloning of protein phosphates and protein kinase genes from crop plants

Protein kinases and protein phosphatases are two very large enzyme families functioning in many important cellular processes such as regulation of cell cycle, signal transduction, self-incompatibility and disease resistance in plants. Some members of these two families have been cloned and subjected to detailed studies, but the majority remains unidentified, especially in plants. Barley and maize are two important crops in the world and also excellent experimental organisms particularly in the area of molecular genetics. Genes encoding protein kinase have been cloned from maize and barley, however, genes encoding protein phosphatase have not been cloned from barley. The signal transduction pathways involving the cloned protein kinases and protein phosphatases in maize and barley are not known. Since the amino acid sequences in the catalytic domains of protein kinase and protein phosphatase is conserved within eukaryotes, our approach consisted of using homology based screening to isolate novel protein kinases from maize cDNA library, and protein phosphatases from barley genomic library