Expression and Characterization of Two Pathogenic Mutations in Human Electron Transfer Flavoprotein

摘要: Defects in electron transfer flavoprotein (ETF) or its acceptor, flavoprotein-ubiquinone oxidoreductase (ETF-QO), cause the human inherited metabolic disease glutaric acidemia type II. In this disease, from nine primary dehydrogenases to main respiratory chain is impaired. Among these are four length-specific of fatty acid beta-oxidation. investigation, two mutations alpha subunit that have been identified patients were expressed Escherichia coli. Of mutant alleles, alphaT266M and alphaG116R, former most frequent mutation found with ETF deficiency. The crystal structure shows alphaG116 lies a hydrophobic pocket, under contact residue alpha/beta interface, hydroxyl hydrogen alphaT266 hydrogen-bonded N(5) FAD; amide backbone C(4)-O flavin prosthetic group (Roberts, D. L., Frerman, F. E. Kim, J-J. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14355-14360). Stable expression alphaG116R required coexpression chaperonins, GroEL GroES. folds into conformation different wild type, catalytically inactive crude extracts. It unstable could not be extensively purified. was purified characterized after stabilization proteolysis Although global protein unchanged, environment altered as indicated by absorption circular dichroism spectroscopy kinetics release oxidized reduced protein. loss bond at binding increase thermodynamic stability semiquinone 10-fold relative ETF. has relatively little effect on reductive half-reaction catalyzed sarcosine medium acyl-CoA which reduce semiquinone. However, kcat/Km ETF-QO coupled acyl-CoA:ubiquinone reductase assay substrate 33-fold; decrease due largest part rate disproportionation ETF-QO.