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

The E1 decarboxylase component of the human branched-chain ketoacid dehydrogenase complex comprises two E1α (45.5 kDa) and two E1β (37.5 kDa) subunits forming an α₂β₂ tetramer. In patients with type IA maple syrup urine disease, the E1α subunit is affected, resulting in the loss of E1 and branched-chain ketoacid dehydrogenase catalytic activities. To study the effect of human E1α missense mutations on E1 subunit assembly, we have developed a pulse-chase labeling protocol based on efficient expression and assembly of human (His)₆-E1α and untagged E1β 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 E1α maple syrup urine disease mutants studied showed aberrant kinetics of assembly with normal E1β in the 2-h chase compared with the wild type and can be classified into four categories of normal (N222S-α and R220W-α), moderately slow (G245R-α), slow (G204S-α, A240P-α, F364C-α, Y368C-α, and Y393N-α), and no (T265R- α) assembly. Prolonged induction in E. coli grown in the YTGK medium or lowering of induction temperature from 37 to 28 °C (in the case of T265R- α), 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 α₂β₂ tetramers. In contrast, a subset of E1α missense mutations caused the occurrence of exclusive αβ dimers (Y393N-α and F364C-α) or of both α₂β₂ tetramers and lower molecular weight species (Y368C-α and T265R-α). Thermal denaturation at 50 °C indicated that mutant E1 proteins aggregated more rapidly than wild type (rate constant, 0.19 min^-1), with the T265R-α mutant E1 most severely affected (rate constant, 4.45 min^-1). The results establish that the human E1α mutations in the putative thiamine pyrophosphate-binding pocket that are studied, with the exception of G204S-α, have no effect on E1 subunit assembly. The T265R-α mutation adversely impacts both E1α 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 α₂β₂ structure.