Project 1. Structure and Function of the Human BCKD Metabolic Machine


The long-term goal of this research project continues to be to understand the molecular and the biochemical mechanism underling the multienzyme complex deficiency in Maple Syrup Urine Disease (MSUD) or branched-chain ketoaciduria. The metabolic disorder is manifested by often-fatal neurological degeneration caused by the accumulation of branched-chain alpha-ketoacids (BCKAs). The multienzyme complex affected in MSUD, the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) complex is a 4-megadalton metabolic machine organized around a 24-meric transacylase (E2b) cubic core, to which multiple copies of a decarboxylase (E1b), a dehydrogenase (E3), a specific kinase and a specific phosphatase are attached through ionic interactions (The BCKD Metabolic Machine). Disease-causing mutations in four (E1b-alpha, E1b-beta, E2b and E3) of the six MSUD loci have been described. Therefore, MSUD provides a genetic model to investigate effects of human mutations on protein-protein interactions and macromolecular assembly. Our laboratory has determined the crystal structure of the human E1b alpha2beta2 heterotetramer at 1.4 to 1.8- resolution (Human E1b structure). The X-ray crystallographic studies have been carried out in collaboration with investigators in the Structural Biology Laboratory in the Biochemistry Department. Based on this new structural information, we propose to:

  1. Characterize the mechanism of ThDP-mediated oxidative decarboxylation at the human E1b active site (Human E1b Active site), and dissect conformational changes in the E1b active site induced by phosphorylation.

  2. Decipher the mechanisms by which MSUD mutations may interfere with hydrophobic-core packing, and subunit interactions in E1, and develop strategies to mitigate these putative folding defects.

  3. Define structural determinants in the binding domain of E2 for interactions with E1 and E3, and determine the three-dimensional structure of the human BCKD complex.


Supported by NIH grant DK-26758 in year 26