ARS | Publication request: Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of Ser292 by pyruvate dehydrogenase-kinase

Submitted to: Enzyme Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 5, 2010
Publication Date: January 20, 2011
Citation: Hirani, T.A., Tovar-Mendez, A., Miernyk, J.A., Randall, D.D. 2011. Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of Ser292 by pyruvate dehydrogenase-kinase. Enzyme Research. 2011:1-13. Article 939068. Available: http://www.sage-hindawi.com/journals/er/.

Interpretive Summary: The use of a specialized chemical method to study components of plants is described. Specifically, the use of this method to study sexual reproduction in seed plants is described in detail. In many instances, results from use of this method have been over or poorly interpreted. These errors are described, along with suggestions about how to avoid making them in future studies. Each stage of sexual reproduction is considered individually, although the emphasis is on studies of seed development or germination. A detailed prospectus is included describing research areas that need more attention in the future. This information will be useful to scientists in their efforts to improve agricultural crop production through both classical breeding and application of biotechnology strategies.

Technical Abstract: We have developed an invitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana a2b2-hetero tetrameric pyruvate dehydrogenase (E1) plus A.thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1a, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in E1a 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 E1a. The Asp295 mutant proteins had decreased Vmax values and increased Km values for pyruvate. We propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the gamma-phosphate from ATP to the Ser residue at regulatory site one of E1a.