Teleost fatty acyl desaturase genes : a comparative study.

Abstract

Marine teleosts, unlike their freshwater counterparts, have a repressed ability to synthesise long chain highly unsaturated fatty acids (HUPA). In competent species, the A6 and A5 fatty acid desaturases are critical in the biosynthetic pathway that produces the HUFA’s arachidonic acid (20:4/z-6; AA), eicosapentaenoic acid (20:5n3; EPA) and docosahexaenoic acid (22:6/z-3; DHA) from the Ci8 polyunsaturated fatty acids (PUFA), linoleic acid (18:2/2-6) and a-linolenic acid (18:3/2-3). The deficiency in HUFA biosynthesis in marine fish is of considerable practical significance because, in consequence, farmed marine species require a dietary source of presynthesised HUFA. This is provided by processed products from “industrial” species of marine fish such as sand eel, sardine, capelin and anchovies which themselves obtain HUFA through the food chain. Indicators suggest that the wild fishery supporting the aquaculture feed industry is unsustainable at current levels of exploitation. This has consequential effects on human health as fish, especially marine fish, are the predominant dietary source of HUFA that are crucial for maintaining cell membrane integrity as well as being central to eicosanoid metabolism. Therefore, the primary aims of this project were to further our understanding of the molecular differences in HUFA biosynthesis between marine and freshwater teleosts. This was achieved by comparing the fatty acid desaturase genes of representative marine and freshwater fish. The desaturases are enzymes involved in the biosynthesis of HUFA from PUFA and have been considered as one of the steps that may be compromised in marine fish. The desaturase genes were studied with a view to relating structural, and potential functional differences with different HUFA synthesis phenotypes. During the course of this project sequences of putative desaturase genes were cloned from two freshwater (zebrafish and carp), two marine (turbot and cod) and one anadromous fish species (Atlantic salmon). Once translated, the protein sequences of all the gene products contained all the necessary domains and motifs shown to be required for efficient desaturase function including an N-terminal cytochrome bs domain, and three catalytically important histidine boxes conserved in all members of the gene family. They all included the variant third histidine box that seems typical of A5 and A6 desaturase genes described to date. All of the protein sequences from the fish species had greatest homology to the mammalian desaturases, specifically the human A6 desaturase. The cDNAs of salmon, carp and zebrafish were functionally characterised in Saccharomyces cerevisiae. Three carp transcripts were sequenced and functionally characterised. Two had no A5 or A6 desaturase activity, while the third efficiently desaturated 18:3/2-3 at the A6 position. Of the two functionally characterised salmon transcripts one had no A5 or A6 activity whereas the third efficiently desaturated 20:4/2-3 at the A5 position. The transcripts that had no desaturase activity were considered either non-functioning alleles or pseudogenes acquired as a result of a genome doubling event. It is believed that other A5/6 like desaturases probably exist for both carp and salmon as salmon is known to have high levels of A6 desaturase activity. However, neither the cod nor the turbot cDNAs were functionally characterised in yeast. The most significant result of the functional characterisation study concerned the zebrafish (Danio rerio). The 1590 bp transcript has close similarity to mammalian A6 desaturase. However, the clone encodes a novel desaturase. When expressed in yeast the zebrafish gene confers the ability to convert 18:2/2-6 and 18:3/2-3 to their corresponding A6 desaturase products, 18:3/2-6 and 18:4/2-3. In addition, it confers the ability to convert 20:3/2-6 and 20:4/2-3 to their A5 desaturase products, 20:4/2-6 and 20:5/2-3, respectively. Therefore, the zebrafish gene encodes a bi-functional enzyme having both A6 and A5 desaturase activity. This was the first report of a functionally characterised desaturase of fish, and, in particular, of a fatty acid desaturase with both A6 and A5 activity. The structure of the primary sequences of the fish desaturases were analysed in relation to function and some interesting and potentially highly significant relationships were discovered. However, it was not possible to determine which residue or residues were responsible for the differing substrate specificities between the transcripts. In summary, the results presented in this thesis indicate that (i) all the fish species used in this study possessed desaturase-like sequences (ii) the zebrafish contains a novel, unique desaturase enzyme with both A6 and A5 desaturase activity (iii) marine fish possess A5/6 desaturase-like transcripts (iv) there is some evidence that fish species that have undergone tetraploidy or recent genome duplication appear to have duplicated genes, possibly pseudogenes and/or non-functioning alleles (v) significant differences in primary structure which may have important consequences for function were observed although unequivocal identification of residues responsible for determining function or specificity was not possible. In conclusion, this study has produced results that not only further our understanding of the fatty acid genes of fish but which also furthered our knowledge of the fatty acid desaturases in general. The data will facilitate studies of how fatty acid desaturase primary structures relate to function. Information from this and other studies will lead to complete knowledge of how sequence and structure contribute to confer substrate specificity and how the fatty acid desaturase gene family has evolved