Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila

• Published in: Biochimica et biophysica acta 2012-08, Vol.1822 (8), p.1284-1292
• Main Authors: Alves, Ema, Henriques, Bárbara J., Rodrigues, João V., Prudêncio, Pedro, Rocha, Hugo, Vilarinho, Laura, Martinho, Rui G., Gomes, Cláudio M.
• Format: Article
• Language: English
• Subjects: Acylcarnitines; Alleles; Amino Acid Sequence; Animals; Binding Sites - genetics; Carnitine - analogs & derivatives; Carnitine - metabolism; Drosophila; Drosophila - genetics; Drosophila - metabolism; Electron-Transferring Flavoproteins - genetics; Electron-Transferring Flavoproteins - metabolism; Flavin-Adenine Dinucleotide - genetics; Flavin-Adenine Dinucleotide - metabolism; Flavins - genetics; Flavins - metabolism; Flavoprotein; Genotype; Inherited metabolic defect; Models, Molecular; Molecular Sequence Data; Multiple Acyl Coenzyme A Dehydrogenase Deficiency - genetics; Multiple Acyl Coenzyme A Dehydrogenase Deficiency - metabolism; Multiple acyl-CoA dehydrogenase deficiency (MADD); Mutation; Phenotype; Rossmann fold
• ISSN: 0925-4439; 0006-3002; 1879-260X
• DOI: 10.1016/j.bbadis.2012.05.003

Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of β-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4, C8 and C12) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a β-strand connected by a short loop to an α-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as a potential model organism for MADD. [Display omitted] ► Fly mutants defective for embryonic cuticle allelic to ETF:QO were identified. ► Three missense mutations identified in ETF:QO (G65E, A68V and S104F). ► Raised acylcarnitine levels in fly embryos as in severe MADD. ► Mutations map at FAD motif within Rossmann fold inactivating enzyme. ► Severe MADD-like phenotype in the fruit fly sets proof of concept of suitability as disease model.