Molecular studies of mammalian branched-chain alpha-keto acid dehydrogenase complexes: domain structures, expression, and inborn errors.

We have cloned cDNAs encoding the E1b-alpha, E1b-beta, and E2b subunits of the bovine and human branched-chain alpha-keto acid complexes. The deduced primary structures indicate that the mammalian E2b contains a lipoyl-bearing, an E3-binding, and an inner core domain that are linked in series by two flexible hinge regions. The observed conservation among E2 proteins in each of the three folded domains strongly suggests that the structural cores of alpha-keto acid dehydrogenase complexes are evolutionarily related. We have expressed bovine pre-E2b in E. coli. The lipoate-free precursor protein is enzymatically active and appears to assemble into a 24-mer structure. Studies with deletion mutants support the proposal that the antigenicity in pre-E2b is associated with the flexible proline-rich hinge region. We have observed five distinct molecular phenotypes in maple syrup urine disease (MSUD) cells, according to the pattern of the branched-chain complex protein subunits and mRNAs present. The results have demonstrated a high degree of genetic heterogeneity in MSUD and have identified the affected genes which must be characterized.