Purification and characterization of recombinant pyruvate dehydrogenase kinases from pea and soybean plants [abstract]

Abstract

The pyruvate dehydrogenase complex (PDC) is a large multienzyme complex catalyzing the oxidative decarboxylation of pyruvate and concomitant reduction of NAD to yield acetyl-CoA and NADH. The plant PDCs have vital roles in catabolic and anabolic metabolism. The plant complexes contain three primary components: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). Additionally, mitochondrial PDC (mtPDC) contains two associated regulatory enzymes: pyruvate dehydrogenase kinase (PDK) and phospho-pyruvate dehydrogenase phosphatase. PDK catalyzes phosphorylation on the  subunit of E1, resulting in inactivation of the complex. We have cloned two PDKs from soybean and recently we have cloned three PDKs from pea. cDNAs encoding soybean PDK 1 and 2 and pea PDK 1, 2 and 3 were subcloned into pET expression vector and E. coli BL21 (DE3) cells were transformed with each pET-28-H6-PDK construct. Recombinant proteins were expressed and purified by Ni-NTA agarose column chromatography to approximately 95% homogeneity. Biochemical characterization of these proteins is underway.

The pyruvate dehydrogenase complex (PDC) is a large multienzyme complex catalyzing the oxidative decarboxylation of pyruvate and concomitant reduction of NAD to yield acetyl-CoA and NADH. The plant PDCs have vital roles in catabolic and anabolic metabolism. The plant complexes contain three primary components: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). Additionally, mitochondrial PDC (mtPDC) contains two associated regulatory enzymes: pyruvate dehydrogenase kinase (PDK) and phospho-pyruvate dehydrogenase phosphatase. PDK catalyzes phosphorylation on the a subunit of E1, resulting in inactivation of the complex. We have cloned two PDKs from soybean and recently we have cloned three PDKs from pea. cDNAs encoding soybean PDK 1 and 2 and pea PDK 1, 2 and 3 were subcloned into pET expression vector and E. coli BL21 (DE3) cells were transformed with each pET-28-H6-PDK construct. Recombinant proteins were expressed and purified by Ni-NTA agarose column chromatography to approximately 95% homogeneity. Biochemical characterization of these proteins is underway.