Pisum sativum mitochondrial pyruvate dehydrogenase can be assembled as a functional alpha(2)beta(2) heterotetramer in the cytoplasm of Pichia pastoris

Pea (Pisum sativum) mitochondrial pyruvate dehydrogenase (E1) was produced by coexpression of the mature alpha and beta subunits in the cytoplasm of t he yeast Pichia pastoris. Size-exclusion chromatography of recombinant E1, using a Superose 12 column, yielded a peak at M-r 160,000 that contained bo th 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 wa s purified by affinity chromatography using immobilized Ni+, with a yield o f 2.8 mg L-1. The pyruvate-decarboxylating activity of recombinant E1 was d ependent upon added Mg2+ and thiamin-pyrophosphate and was enhanced by the oxidant potassium ferricyanide, Native pea mitochondrial E1-kinase catalyze d phosphorylation of Ser residues in the alpha-subunit of recombinant E1, w ith concomitant loss of enzymatic activity. Thus, mitochondrial pyruvate de hydrogenase can be assembled in the cytoplasm of P. pastoris into an alpha( 2)beta(2) heterotetramer that is both catalytically active and competent fo r regulatory phosphorylation. (C) 2000 Academic Press.