Maple syrup urine disease (MSUD) is an autosomal recessive disorder in the oxidative decarboxylation of the branched-chain alpha-keto acids derived from leucine, isoleucine and valine. The enzyme deficient in MSUD, the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex, is a mitochondrial multienzyme complex consisting of at least six distinct subunits. MSUD is genetically heterogeneous as manifested by lesions in different subunits of the BCKAD complex among unrelated patients. To approach the biochemical basis of MSUD involving the dihydrolipoyl transacylase (E2) subunit, the domain structure of this polypeptide from human and bovine livers has been defined by limited proteolysis and cDNA cloning. The assembly of 24 E2 subunits into a cubic structure, forming the core of the mammalian BCKAD complex, was established by electron microscopy and sedimentation equilibrium analysis. Highly assembled bovine E2 devoid of prosthetic lipoic acid has been overexpressed in Escherichia coli. Studies carried out with this bacterial expression system have provided insights into the lipoylation process of E2, and the involvement of the His391 residue in the transacylation reaction. At the genetic level, the human E2 gene (DBT) has been regionally assigned to chromosome 1p31, and a related E2 pseudogene to chromosome 3q24 by in situ hybridization. Genomic cloning has shown that the human E2 gene undergoes premature transcriptional termination and alternate splicing as normal events, although its functional significance is unknown. Through the use of the polymerase chain reaction and other recombinant DNA methods, several compound heterozygous mutations at the E2 locus have been identified in classical as well as thiamine-responsive MSUD patients. These mutations would appear to be useful genetic models, which will facilitate investigations into macromolecular organization and protein-protein interactions. Moreover, an array of precise single and multiple exon deletions has been observed in the amplified mutant E2 transcripts. The results represent unexpected secondary effects that are apparently associated with the above primary mutations in the E2 gene.