A vertebrate fatty acid desaturase with Delta 5 and Delta 6 activities

Abstract

Delta5 and Delta6 fatty acid desaturases are critical enzymes in the pathways for the biosynthesis of the polyunsaturated fatty acids arachidonic, eicosapentaenoic, and docosahexaenoic acids. They are encoded by distinct genes in mammals and Caenorhabditis elegans. This paper describes a cDNA isolated from zebrafish (Danio rerio) with high similarity to mammalian Delta6 desaturase genes. The 1,590-bp sequence specifies a protein that, in common with other fatty acid desaturases, contains an N-terminal cytochrome b(5) domain and three histidine boxes, believed to be involved in catalysis. When the zebrafish cDNA was expressed in Saccharomyces cerevisiae it conferred the ability to convert linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) to their corresponding Delta6 desaturated products, 18:3n-6 and 18:4n-3. However, in addition it conferred on the yeast the ability to convert di-homo-gamma-linoleic acid (20:3n-6) and eicosatetraenoic acid (20:4n-3) to arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3), respectively, indicating that the zebrafish gene encodes an enzyme having both Delta5 and Delta6 desaturase activity. The zebrafish Delta5/Delta6 desaturase may represent a component of a prototypic vertebrate polyunsaturated fatty acids biosynthesis pathway.

Figure 1

Alignment of the predicted amino acid sequences of the zebrafish desaturase (DRD5/6) and human Δ5 (HSFADS1) and Δ6 desaturases (HSFADS2). The three ORFs encode 444 amino acid residues. Identical amino acids are in black, the cytochrome b₅-like domain is underlined by asterisks, and the three histidine-rich domains are underlined.

Figure 2

Phylogeny of desaturase deduced amino acid sequences. Sequences marked with an asterisk are not functionally characterized. Database accession numbers for the nucleic acid sequences are indicated. The conditions used for neighbor-joining tree construction are described in Materials and Methods and were applied by using CLUSTALW and NJPLOT. Horizontal branch lengths are proportional to the number of amino acid replacements per position, and the scale bar indicates this value. Numbers represent the percentage frequencies with which the tree topology presented here was replicated after 1,000 bootstrap iterations.

Figure 3

Identification of fatty acid desaturation products in transgenic yeast. FAMEs were extracted from yeast transformed with pYESZFB10 grown in the presence of 18:2n-6 (A), 18:3n-3 (B), 20:3n-6 (C), or 20:4n-3 (D). The first four peaks in A–D are 16:0, 16:1n-7, 18:0, and 18:1n-9, respectively. The fifth peaks in each panel are the exogenously added fatty acids 18:2n-6 (A), 18:3n-3 (B), 20:3n-6 (C), and 20:4n-3 (D), respectively. The sixth peaks in each panel (arrowed) were identified tentatively (based on retention times) as 18:3n-6 (A), 18:4n-3 (B), 20:4n-6 (C), and 20:5n-3 (D), respectively.

Figure 4

Mass spectra of the arrowed peaks in Fig. 3. Picolinyl esters were prepared from FAMEs extracted from yeast transformed with pYESZFB10 grown in the presence of 18:2n-6 (A), 18:3n-3 (B), 20:3n-6 (C), and 20:4n-3 (D) and analyzed by GC-MS as described in Materials and Methods. The identities of the peaks were confirmed as 18:3n-6 (A), 18:5n-3 (B), 20:4n-6 (C), 20:5n-3 (D).