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Title: “Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

item ASHAN, NAGIB - University Of Missouri
item SWATEK, KIRBY - University Of Missouri
item ZHANG, JINGFEN - University Of Missouri
item Miernyk, Jan
item XXU, DOUG - University Of Missouri
item THELEN, JAY - University Of Missouri

Submitted to: Frontiers in Plant Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/18/2012
Publication Date: 7/16/2012
Citation: Ashan, N., Swatek, K.N., Zhang, J., Miernyk, J.A., Xxu, D., Thelen, J.J. 2012. “Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex. Frontiers in Plant Proteomics. 3:1-10.

Interpretive Summary: Respiration is the use of energy to do work. The rate of respiration must be carefully controlled so that energy is not wasted. Respiration can be controlled by chemical modification of a key enzyme. A method was developed that allows testing of other parts of the enzyme affect the rate of chemical modifications. The results indicate that the structure of the enzyme can tolerate surprisingly few changes without disturbing the control ability. This information will be useful to scientists who are trying to develop both biotechnology and traditional strategies to improve crop plant productivity through control of respiration.

Technical Abstract: The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1alpha subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client assay plus recombinant pyruvate dehydrogenase E1alpha and E1alpha-kinase to perform “scanning mutagenesis” of the residues flanking the site of phosphorylation. Consistent with the results from “phylogenetic analysis” of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.