Role of Elongase Reactions in LCPUFA Synthesis Figure 2. Competition between n-3 PUFA substrates for elongation by Elovl5. Recombinant S. cerevisiae expressing Elovl5 were grown in the presence of 100 mM of SDA and 0–300 mM EPA (A) or 100 mM of EPA and 0–300 mM SDA (B). Recombinant S. cerevisiae containing the empty pYES2 vector were grown in the presence of 0–300 mM EPA or SDA. Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was quantified in mg g21 of S. cerevisiae or expressed as a percentage of the total amount of all fatty acids. The proportion of substrate fatty acid converted to longer chain fatty acid product(s) was calculated as [product(s)/(product(s) + substrate)]6100. The conversion of SDA includes the 4 carbon elongation product 22:4n-3. The results are the means 6 S.D. of triplicate incubations. Values with different symbols are significantly different from each other. doi:10.1371/journal.pone.0029662.g002 acyl desaturases; the conserved histidine boxes or cytochrome b5 domain containing the heme-binding motif. Fads2 substrate specificities. Recombinant S. cerevisiae cells expressing Fads2 were cultured in the presence of 100 mM of various C18-24 PUFA substrates to determine the enzymes substrate specificity. Fads2 demonstrated D6-desaturase activity with the expected n-3 and n-6 substrates, ALA and LA, and with 24:5n-3 (Fig. 5). Fads2 did not exhibit D5-desaturase activity with the substrates 20:4n-3 or 20:3n-6, or D8-desaturase activity with the substrates 20:3n-3 and 20:2n-6 (data not shown). Competition between n-3 C18 and C24 substrates for Fads2. ALA and 24:5n-3 were both Fads2 substrates with similar conversion efficiencies at a single concentration (Fig. 5). Therefore, the potential for competitive inhibition was examined at concentrations of ALA and 24:5n-3 that were in the linear response range (Fig. 6). The concentration of 24:5n-3 that was examined was much lower than the concentration of ALA. However, 24:5n-3 is likely to be lower than ALA in vivo because 24:5n-3 is usually not detectable in ALA dietary studies. Increasing concentrations of ALA over the range 0–400 mM dose- dependently inhibited the conversion of 24:5n-3 to 24:6n-3 (Fig. 7). Discussion Our direct comparative study of the rat elongases showed that the substrates for Elovl5 are C18 and C20 PUFA, while for Elovl2 they are C20 and C22 PUFA (Fig. 8). This is generally concordant with specificities from separate studies of mouse Elovl2 [6], human Elovl2 [6], human Elovl5 [4] and rat Elovl5 [5], although there are some differences. The rELO1 (now Elovl5) examined by Inagaki et al. [5] had four residue differences from the Elovl5 examined in this study and it had markedly greater preferences for C18 over C20 substrates, both n-3 and n-6, which were not observed in our study. Also, compared with the rat Elovl2 in our study, the mouse Elovl2 appeared to convert EPA preferentially through to 24:5n-3 with less accumulation of DPA [6]. However, only a single concentration of EPA was used in the mouse study. We have shown that the proportion of EPA converted to 24:5n-3 by Elovl2 compared with that accumulating as DPA, diminishes with increasing EPA concentration. A possible explanation is that the second reaction, DPA to 24:5n-3, becomes saturated. We also examined competitive substrate interactions. Elovl2 and Elovl5 each have at least two n-3 substrates, raising the PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29662