Functional analysis in Saccharomyces cerevisiae of naturally occurring amino acid substitutions in human dihydrolipoamide dehydrogenase.
Dihydrolipoamide dehydrogenase is a common component of mammalian multienzyme complexes that decarboxylate alpha-ketoacids and catabolize glycine. The common function is to reoxidize a reduced lipoate component of each complex, thereby preparing that lipoate for another round of catalysis. Regions within dihydrolipoamide dehydrogenase involved in association with other proteins of the complexes are poorly defined, and despite high amino acid sequence conservation through evolution, it is unknown if dihydrolipoamide dehydrogenases are functionally equivalent across species. To address this issue, we asked whether the human enzyme could restore function to the alpha-ketoacid dehydrogenase complexes in a yeast strain deficient in endogenous dihydrolipoamide dehydrogenase. This dihydrolipoamide dehydrogenase null mutant will not grow on non-fermentable carbon sources. The human enzyme expressed from a CEN plasmid complemented the growth phenotype and restored full activity to the pyruvate and alpha-ketoglutarate dehydrogenase complexes. Human dihydrolipoamide dehydrogenases with selected amino acid substitutions were then tested in the null strain for their ability to restore function. Substitutions tested represented naturally occurring candidate mutations identified in an individual with inactive dihydrolipoamide dehydrogenase. A K37E change had full function while a P453L change resulted in reduced dihydrolipoamide dehydrogenase abundance in the mitochondria and no detectable catalytic activity.
Department of Genetics and Molecular Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
M M
MM
Lanterman
J R
JR
Dickinson
D J
DJ
Danner
D000596:Amino Acids / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics*; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000596:Amino Acids; D008058:Dihydrolipoamide Dehydrogenase