Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl d ehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzym e complex from Bacillus stearothermophilus were overexpressed in Escherichi a coli, The E2 component was purified as a large soluble aggregate (molecul ar mass >1x10(6) Dal with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa, The recombinant E2 component in vitro was capable of binding either 6 0 E3(alpha(2)) dimers or 60 heterotetramers (alpha(2)beta(2)) of the pyruva te decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into t he icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has i mportant consequences for the stoichiometry of the assembled complex in viv o. The lipoyl domain of the recombinant E2 protein was found to be unlipoyl ated, but it could be correctly post-translationally modified in vitro usin g a recombinant lipoate protein ligase from E, coli. The lipoylated E2 comp onent was able to bind recombinant E1 and E3 components in vitro to generat e a PDH complex with a catalytic activity comparable with that of the wild- type enzyme. Reversible unfolding of the recombinant E2 and E3 components i n 6 M guanidine hydrochloride was possible in the absence of chaperonins, w ith recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the El enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1 alpha and E1 beta. Thi s represents the first complete post-translational modification and assembl y of a fully active PDH complex from recombinant proteins in vitro.