Impaired assembly of E1 decarboxylase of the branched-chain alpha-ketoacid dehydrogenase complex in type IA maple syrup urine disease.

The E1 decarboxylase component of the human branched-chain ketoacid dehydrogenase complex comprises two E1alpha (45.5 kDa) and two E1beta (37.5 kDa) subunits forming an alpha2 beta2 tetramer. In patients with type IA maple syrup urine disease, the E1alpha subunit is affected, resulting in the loss of E1 and branched-chain ketoacid dehydrogenase catalytic activities. To study the effect of human E1alpha missense mutations on E1 subunit assembly, we have developed a pulse-chase labeling protocol based on efficient expression and assembly of human (His)6-E1alpha and untagged E1beta subunits in Escherichia coli in the presence of overexpressed chaperonins GroEL and GroES. Assembly of the two 35S-labeled E1 subunits was indicated by their co-extraction with Ni2+-nitrilotriacetic acid resin. The nine E1alpha maple syrup urine disease mutants studied showed aberrant kinetics of assembly with normal E1beta in the 2-h chase compared with the wild type and can be classified into four categories of normal (N222S-alpha and R220W-alpha), moderately slow (G245R-alpha), slow (G204S-alpha, A240P-alpha, F364C-alpha, Y368C-alpha, and Y393N-alpha), and no (T265R-alpha) assembly. Prolonged induction in E. coli grown in the YTGK medium or lowering of induction temperature from 37 to 28 degreesC (in the case of T265R-alpha), however, resulted in the production of mutant E1 proteins. Separation of purified E1 proteins by sucrose density gradient centrifugation showed that the wild-type E1 existed entirely as alpha2 beta2 tetramers. In contrast, a subset of E1alpha missense mutations caused the occurrence of exclusive alphabeta dimers (Y393N-alpha and F364C-alpha) or of both alpha2beta2 tetramers and lower molecular weight species (Y368C-alpha and T265R-alpha). Thermal denaturation at 50 degreesC indicated that mutant E1 proteins aggregated more rapidly than wild type (rate constant, 0.19 min-1), with the T265R-alpha mutant E1 most severely affected (rate constant, 4.45 min-1). The results establish that the human E1alpha mutations in the putative thiamine pyrophosphate-binding pocket that are studied, with the exception of G204S-alpha, have no effect on E1 subunit assembly. The T265R-alpha mutation adversely impacts both E1alpha folding and subunit interactions. The mutations involving the C-terminal aromatic residues impede both the kinetics of subunit assembly and the formation of the native alpha2 beta2 structure.