Biochemical basis of type IB (E1 beta) mutations in maple syrup urine disease - A prevalent allele in patients from the Druze kindred in Israel

Maple syrup urine disease (MSUD) is a metabolic disorder associated with of ten-fatal ketoacidosis, neurological derangement, and mental retardation. I n this study, we identify and characterize two novel type ID MSUD mutations in Israeli patients, which affect the E1 beta subunit in the decarboxylase (El) component of the branched-chain a-ketoacid dehydrogenase complex. The recombinant mutant El carrying the prevalent S289L-beta (TCG --> TTG) muta tion in the Druze kindred exists as a stable inactive alpha beta heterodime r. Based on the human El structure, the S289L-beta mutation disrupts the in teractions between Ser-289-beta and Glu-290-beta', and between Arg-309-beta and Glu-290-beta', which are essential for native alpha (2)beta (2) hetero tetrameric assembly. The R133P-beta (CGG --> CCG) mutation, on the other ha nd, is inefficiently expressed in Escherichia coli as heterotetramers in a temperature-dependent manner. The R133P-beta mutant El exhibits significant residual activity but is markedly less stable than the wild-type, as measu red by thermal inactivation and free energy change of denaturation. The R13 3P-beta substitution abrogates the coordination of Arg-133-beta to Ala-95-b eta, Glu-96-beta, and Ile-97-beta, which is important for strand-strand int eractions and K+ ion binding in the beta subunit. These findings provide ne w insights into folding and assembly of human El and will facilitate DNA-ba sed diagnosis for MSUD in the Israeli population.