Defects in electron transfer flavoprotein (ETF) result in glutaric acidemia type II (GA2), a human inborn error of fatty acid and amino acid metabolism. The objective of this research is to establish the molecular bases, at the nucleotide and protein levels, of ETF deficiency. To achieve this goal we shall develop a full length cDNA clone encoding the human beta subunit and use this cDNA, along with the cDNA encoding the alpha ETF subunit which we already have, to map mutations in the human genes. The authenticity of the beta ETF cDNA will be established by expressing the cDNA in a human fibroblast line deficient in the beta subunit. The effects of the human mutations on catalysis and chemistry of the flavin prosthetic group will be investigated by expressing the mutations in the ETF of Paracoccus denitrificans, after site directed mutagenesis of the bacterial genes. The bacterial ETF is structurally, catalytically and immunologically very similar to human ETF. This approach will be used because a system for simultaneous expression of nonidentical subunits of eucaryotic proteins from cDNA has not been developed. Analyses of the mutant proteins may impact on treatment of GA2 patients with partial ETF deficiency and will enhance understanding of ETF structure/function relationships.
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