Submitted to: Biochemical Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/19/2000
Publication Date: 5/1/2000
Citation: Thelen, J.J., Miernyk, J.A., Randall, D.D. 2000. Pyruvate dehydrogenase kinase from arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues. Biochemical Journal. V.349:195-201. Interpretive Summary: Respiration is the use of energy by living cells to do work. Both growth and reproduction are affected by respiration. As a result, respiration must be carefully controlled or wasted energy will decrease crop yields and reduce agricultural productivity. The control of respiration in plant cells is a subject of ongoing study. A protein that is important in the regulation of respiration was isolated from the model plant thale cress an studied. Comparisons were made with a similar protein from micro-organisms in order to predict characteristics that might be important in control of respiration. A method was developed to produce large amounts of the plant protein in the laboratory for chemical analysis. An additional method was developed to induce a change in the chemical composition of the plant protein, and to test if the change had any effect upon the proposed function. This information will be important to researchers in their attempts to increase agricultural productivity by altering the control of plant cell respiration, and to other plant scientists who will try to design more efficient crop plants through either classical plant breeding or biotechnology.
Technical Abstract: The pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex. Although PDKs inactivate the pyruvate dehydrogenase complex by phosphorylating specific Ser residues, the primary amino acid sequence indicates they are descendants of prokaryotic histidine kinases rather than eukaryotic Ser/Thr rkinases. Unlike Ser/Thr kinases, histidine kinases utilize a conserved His for phosphotransfer to Asp residues. To better understand these unique kinases, we have begun molecular analysis of the reaction mechanism. A presumptive PDK from Arabidopsis thaliana was expressed and purified from Escherichia coli. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial pyruvate dehydrogenase complex by phosphorylating Ser residues. Additionally, A. thaliana PDK is capable of autophosphorylating Ser residues near its amino-terminus, though this phospho-Ser is not a phosphotransfer intermediate. To elucidate the mechanism for phosphotransfer, we performed site-directed mutagenesis of the His residue purported to be involved in phosphotransfer. When H121 was mutated to Ala or G1n, Ser-autophosphorylation was reduced by 50%. Trans- phosphorylation of the pyruvate dehydrogenase complex was concomitantly reduced. It is postulated that either H121 is not the sole phosphotransfer phosphotransfer His residue, or that mutagenesis of H121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus, H121 is one residue involved in intermolecular phospho-relay, and PDK is the first example of a protein histidine kinase that both autophosphorylates and trans-phosphorylates Ser residues.