Deciphering the role of cytochrome b5-desaturase interactions for fatty acid desaturation.

摘要

Very long chain polyunsaturated fatty acids (VLC-PUFAs) including arachidonic acid and eicosapentaenoic acid are important in human health and development. Humans cannot synthesize these fatty acids de novo therefore must obtain them from other sources. Fish oils have become a valuable source of VLC-PUFAs. However, with fish populations in decline, new sustainable sources of essential fatty acids must be developed. The production of VLC-PUFAs depends on metabolic pathways including the desaturation electron transport chain. Fatty acid desaturation is composed of three electron transfer partners: desaturase, cytochrome b5, and cytochrome b5 reductase. The limiting step in VLC-PUFA production is the Delta6 desaturase. The Delta6 desaturase from the ciliated protist, Tetrahymena thermophila (TtDes6) was characterized by heterologous expression in yeast. When yeast cells were supplemented with 18:2Delta9,12, 4% is desaturated into 18:3Delta6,9,12. However, when coexpressed with cytochrome b5 proteins (TtCB5-A and TtCB5-B) identified from the genome of Tetrahymena thermophila, desaturation increased to 40% and 25% respectively. The cytochrome b5 from A. thaliana was also shown to increase desaturation to 40% when coexpressed with TtDes6. The increased desaturation observed with the addition of cytochrome b5s from T. thermophila and Arabidopsis illustrates the importance of the interaction between cytochrome b5s and desaturases in the synthesis of polyunsaturated fatty acids in heterologous systems. The split ubiquitin assay was used to determine if the physical interaction between cytochrome b5 and desaturase played a role in the differing desaturase activities. This assay indicated that the TtCB5-A interacts with TtDes6 strongly, while the interaction between ScCB5 and TtDes6 appears to be a very weak interaction. This indicates that physical interaction between electron transfer partners plays a vital role in fatty acid desaturase activity. Delta12 desaturase is also vital in the synthesis of essential fatty acids by converting oleic acid to linoleic acid. The Delta12 desaturase from T. thermophila was also characterized. In order to functionally characterize TtDes12 in the heterologous yeast system, a cytochrome b5 from T. thermophila was also required. The goal to optimize novel and unusual fatty acid biosynthesis in heterologous system depends on the ability of newly introduced proteins to interact with the host's proteins. To increase production of target fatty acids in heterologous systems the interactions between proteins involved must be better understood.