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

R. Max Wynn, James R. Davie, Jacinta L. Chuang, Cynthia D. Cote, David T. Chuang

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34 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)13110-13118
Number of pages9
JournalJournal of Biological Chemistry
Issue number21
StatePublished - May 22 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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