Drosophila FAS II & ß-oxidation enzymes

The project described herein is concerned with identifying Drosophila melanogaster genes encoding enzymes for compartmentalised fatty acid metabolism. This three-year project will be based on functional complementation of Saccharomyces cerevisiae fatty acid synthesis or ß-oxidation mutants, and comprise the basis for future studies aimed at characterising fatty acid metabolism in the fruit fly. These envisioned studies might help to elucidate the potential role of peroxisomes and mitochondria, with entrained biochemical pathways, in neuron maintenance and degeneration.

In eukaryotes, fatty acid ß-oxidation as well as type II fatty acid biosynthesis (FAS II) are both compartmentalised pathways. The complete retinue of fungal genes and enzymes engaged in these processes has been published previously using Saccharomyces cerevisiae mutants devoid of the respective functions. However, unlike the situation in yeast, where the former process is solely peroxisomal and the latter takes place only in the mitochondria, the localisation of these pathways in animal cells is somewhat less distinct. Peroxisomes in animal cells contain a ß-oxidation process, but contrary to yeast cells, animal mitochondria accommodate both fatty acid biosynthetic and breakdown pathways. At the onset of this project, no studies had been reported on the effects in multi-cellular organisms of lesions specific to mitochondrial FAS II. Hence, this prompted the identification of genes encoding enzymes of compartmentalised fatty acid metabolism in the fruit fly Drosophila melanogaster so as to determine the consequences to animals of a dysfunctional fatty acid metabolism. Here, the fruit fly genome was scanned for open reading frames similar to known fungal ß-oxidation or FAS II genes that could potentially encode enzymes entrained in fatty acid metabolism, with an emphasis on FAS II. This revealed a number of fruit fly candidates with significant homology to their yeast counterparts. Several fruit fly enzymes were expressed in the mitochondria of yeast mutants lacking the cogent activity, and this identified four fruit fly enzymes capable of functionally replacing the missing yeast proteins. Enzyme activity measurements using transformed yeast cells confirmed the identity of the novel fruit fly enzymes. In collaboration with two specialist laboratories, Drosophila mutants were generated that were designed to be dysfunctional in one of several FAS II or ß-oxidation enzymes, but these flies failed to reveal a specific mutant phenotype. Hence, subsequent efforts were funnelled towards examining the consequences of perturbing FAS II in another metazoan, the nematode Caenorhabitis elegans. The nematode genome was similarly scanned for genes that could encode FAS II enzymes, and this exposed two such genes, F09E10.3 and W09H1.5. The latter gene was experimentally silenced in a dedicated worm laboratory, revealing for the first time that FAS II is linked to longevity in C. elegans. In addition, novel genes coding for fatty acid biosynthesis enzymes were investigated in a number of disease- carrying agents, including Mycobacteria tuberculosis and Leishmania major. This revealed the identity of mycobacterial FabG4 as a 3-oxoacyl-thioester reductase as well as the ability of mycobacterial FabD to replace the relevant Mct1p protein in yeast. Moreover, the Leishmania proteins LmjF07.0430/LmjF07.0440 and LmjF27.2440 could replace the yeast enzymes Htd2p or Oar1p, respectively.