title: Pisum sativum mitochondrial pyruvate dehydrogenase can be assembled as a functional alpha(2)beta(2) heterotetramer in the cytoplasm of Pichia pastoris.
abstract: Pea (Pisum sativum) mitochondrial pyruvate dehydrogenase (E1) was produced by coexpression of the mature alpha and beta subunits in the cytoplasm of the yeast Pichia pastoris. Size-exclusion chromatography of recombinant E1, using a Superose 12 column, yielded a peak at M(r) 160,000 that contained both alpha and beta subunits as well as E1 activity. This corresponds to the size of native alpha(2)beta(2) E1. Recombinant E1 alpha (His(6))-E1 beta was purified by affinity chromatography using immobilized Ni(+), with a yield of 2.8 mg L(-1). The pyruvate-decarboxylating activity of recombinant E1 was dependent upon added Mg(2+) and thiamin-pyrophosphate and was enhanced by the oxidant potassium ferricyanide. Native pea mitochondrial E1-kinase catalyzed phosphorylation of Ser residues in the alpha-subunit of recombinant E1, with concomitant loss of enzymatic activity. Thus, mitochondrial pyruvate dehydrogenase can be assembled in the cytoplasm of P. pastoris into an alpha(2)beta(2) heterotetramer that is both catalytically active and competent for regulatory phosphorylation.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, Missouri, 65211, USA.
authors.forename: J I
authors.initials: JI
authors.lastname: Moreno
authors.forename: N R
authors.initials: NR
authors.lastname: David
authors.forename: J A
authors.initials: JA
authors.lastname: Miernyk
authors.forename: D D
authors.initials: DD
authors.lastname: Randall
mesh_terms: D001345:Autoradiography; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000201:enzymology*; D008928:Mitochondria / Q000737:chemistry*; D018532:Pisum sativum / Q000737:chemistry* / Q000648:ultrastructure; D010766:Phosphorylation; D010843:Pichia / Q000201:enzymology* / Q000648:ultrastructure; D017510:Protein Folding; D030481:Pyruvate Dehydrogenase (Lipoamide); D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D030481:Pyruvate Dehydrogenase (Lipoamide)