Fatty acyl-CoA reductases of birds

Abstract

Background: Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters.

Results: cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland.

Conclusion: The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.

Figure 1

Evolutionary relationships of FAR from different taxa. Amino acid sequences of FAR from birds (Anser anser domesticus, Ad; Gallus gallus domesticus, Gg; Tyto alba, Ta), mammals (Homo sapiens, Hs; Mus musculus, Mm), insects (Apis mellifera, Am; Yponomeuta evonymellus, Ye; Yponomeuta rorellus, Yr; Yponomeuta padellus, Yp; Ostrinia nubilalis, On; Ostrinia scapulalis, Os; Bombyx mori, Bm), protozoa (Euglena gracilis, Eg) and plants (Arabidopsis thaliana, At; Triticum aestivum L., Ta; Simmondsia chinensis, Sc) were compared to calculate a bootstrap consensus tree. Numbers above branches: bootstrap values, scale: number of amino acid differences per site.

Figure 2

Alcohol production of yeast strains expressing avian FAR enzymes from endogenous fatty acids. (a) TLC analysis of lipid extracts from transgenic yeast cells. Total lipid fraction of yeast cells expressing one of the avian FAR sequences or the empty vector control were extracted after 72 h incubation and lipid extracts were analyzed by TLC. FFA: free fatty acids, R-OH: fatty alcohols. (b) Alcohol composition of transgenic yeast strains. The alcohol composition was analyzed and quantified by GC. Results are mean values of duplicate extractions from two independent yeast cultures. Following concentrations in μmol*g^-1 fresh weight correspond to 100%: AdFAR1:15.6, GgFAR1:13.2, TaFAR1: 17.7, GgFAR2: 8.2, TaFAR2: 2.9, control: 0.02. Because GgFAR1db showed results very similar to those of GgFAR1, results of GgFAR1 are presented only.

Figure 3

Alcohol production of yeast strains expressing avian FAR enzymes supplemented with a mixture of odd-numbered saturated fatty acids. Yeast cells expressing one of the avian FAR proteins or the empty vector control were supplemented with a mixture of 13:0, 15:0, 17:0, 19:0, extracted after 72 h incubation and synthesized fatty alcohols were analyzed by GC. Results are mean values of extractions from at least three independent yeast cultures. Following alcohol concentrations in μmol*g^-1 fresh weight correspond to 100%: AdFAR1: 16.1, GgFAR1: 10.6, TaFAR1: 15.8, GgFAR2: 15.6, TaFAR2: 3.9, control: 0.0.

Figure 4

Cofactor requirement of FAR enzymes. (a) Cofactor requirement of TaFAR1 and TaFAR2. Assays were carried out with the total membrane fraction of yeast cells expressing TaFAR1 or TaFAR2. [1-¹⁴C]-labeled products derived from assays carried out with labeled 16:0-CoA (lane 1 and 2, TaFAR1) or labeled 18:0-CoA (lane 3 and 4, TaFAR2) and NADH (lane 1 and 3) or NADPH (lane 2 and 4). (ALD: fatty aldehyde, FFA: free fatty acid, R-OH: fatty alcohol). (b) NADPH dependency of TaFAR1. Assays were conducted with the total membrane fraction of yeast cells expressing TaFAR1 and with [1-¹⁴C]-labeled 16:0-CoA. The concentration of NADPH was varied under otherwise standard assay conditions. Results are the sum of synthesized aldehydes and alcohols and display mean values of two independent assays.

Figure 5

Acyl-CoA dependencies of TaFAR1 (a) and TaFAR2 (b). The total membrane fractions of yeast cells expressing TaFAR1 or TaFAR2 were used as enzyme source. Different concentrations of the given [1-¹⁴C]-labeled acyl-CoA species were tested under optimal assay conditions. Results are the sum of synthesized aldehydes and alcohols and display mean values of two independent assays.

Figure 6

Substrate specificities of avian FAR enzymes. In vitro assays were conducted with the total membrane fractions of yeast cells harboring one of the avian FAR sequences under optimal conditions and with [1-¹⁴C]-labeled acyl-CoA thioesters. Results are mean values of assays conducted in duplicate with three independent membrane preparations and represent the sum of synthesized alcohols and aldehydes. 100% correspond to the following FAR activities in nmol*min^-1*mg^-1 membrane protein: AdFAR1: 1.7, GgFAR1: 8.2, TaFAR1: 9.2, GgFAR2: 3.7, TaFAR2: 2.6, control: 0.01.

Figure 7

(a) Activities of avian FAR enzymes with branched-chain acyl-CoA thioesters. The total membrane fractions of yeast cells harboring one of the avian FAR sequences were used as enzyme source and [1-¹⁴C]-labeled 2-methyl-branched acyl-CoA thioesters as substrate. Results are mean values of assays conducted in duplicate with three independent membrane preparations and represent the sum of synthesized alcohols and aldehydes. 100% correspond to the following FAR activities in pmol*min^-1*mg^-1 protein: AdFAR1: 25, GgFAR1: 170, TaFAR1: 200, GgFAR2: 60, TaFAR2: 18, control: 10. (b) TLC analysis of FAR assay products with branched-chain acyl-CoA thioesters. Reaction products of assays with yeast membranes harboring either GgFAR1 or GgFAR2 are shown.

Figure 8

Semi-quantitative expression analyses of GgFAR1 and GgFAR2 sequences in chicken tissues. Partial cDNA sequences were amplified by RT-PCR using 1 μg of total RNA isolated from different tissues as template. Synthesis of partial β-actin cDNA was carried out as a control for RNA load. By reactions without reverse transcriptase DNA contamination was excluded.