FAD insertion is essential for attaining the assembly competence of the dihydrolipoamide dehydrogenase (E3) monomer from Escherichia coli

Dihydrolipoamide dehydrogenase (E3) from Escherichia coli, an FAD-linked ho modimer, can be fully reconstituted in. vitro following denaturation in 6 M guanidinium chloride. Complete restoration of activity occurs within 1-2 h in the presence of FAD, dithiothreitol, and bovine serum albumin. In the a bsence of FAD, the dihydrolipoamide dehydrogenase monomer forms a stable fo lding intermediate, which is incapable of dimerization, This intermediate d isplays a similar tryptic resistance to the native enzyme but is less heat- stable, because its ability to form native E3 is lost after incubation at 6 5 degreesC for 15 min, The presence of FAD promotes slow, additional confor mational rearrangements of the E3 subunit as observed by cofactor-dependent decreases in intrinsic tryptophan fluorescence. However, after 2 h, the tr yptophan fluorescence spectrum and far UV CD spectrum of E3, refolded in th e absence of FAD, are similar to that of the native enzyme, and full activi ty can still be recovered on addition of FAD, Cross-linking studies show th at FAD insertion is necessary for the monomeric folding intermediate to att ain an assembly competent state leading to dimerization. Thus cofactor inse rtion represents a key step in the assembly of this enzyme, although its in itial presence appears not to be required to promote the correct folding pa thway.