Elongase Reactions as Control Points in Long-Chain Polyunsaturated Fatty Acid Synthesis Melissa K. Gregory1*, Robert A. Gibson2, Rebecca J. Cook-Johnson1, Leslie G. Cleland 1 , Michael J. James1 1 Rheumatology Unit, Royal Adelaide Hospital, Adelaide, Australia, 2 Food Science, School of Agriculture, Food and Wine, University of Adelaide, Adelaide, Australia Abstract Background: D6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary a-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA. Methodology/Principal Findings: The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C20 and C22 polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPARDPAR24:5n-3. The second reaction DPAR24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPARDPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3. Conclusions: The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPAR24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means. Citation: Gregory MK, Gibson RA, Cook-Johnson RJ, Cleland LG, James MJ (2011) Elongase Reactions as Control Points in Long-Chain Polyunsaturated Fatty Acid Synthesis. PLoS ONE 6(12): e29662. doi:10.1371/journal.pone.0029662 Editor: Matej Oresic, Governmental Technical Research Centre of Finland, Finland Received July 26, 2011; Accepted December 2, 2011; Published December 22, 2011 Copyright: 2011 Gregory et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Health and Medical Research Council of Australia and the Australian Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: melissa.gregory@health.sa.gov.au Introduction Metabolism of the vegetable-derived omega-3 (n-3) fatty acid, a- linolenic acid (18:3n-3; ALA), to the long-chain n-3 polyunsatu- rated fatty acids (PUFA), eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA), requires progressive desaturation and elongation. The location of regulatory steps in the pathway has largely been inferred from the intermediate fatty acids that accumulate in tissues following increased exposure to ALA. With increasing abundance of ALA or stearidonic acid (18:4n-3; SDA), which is the direct D6-desaturase (Fads2) product of ALA desaturation, the next metabolite to accumulate is EPA [1,2,3]. Although it is commonly reported that Fads2 is rate- limiting for the metabolism of ALA to DHA, the accumulation of EPA and not DHA suggests other control points need to be considered [1,2,3]. As EPA increases, the next metabolite to accumulate is the elongation product docosapentaenoic acid (22:5n-3, DPA) [3]. The accumulation of EPA and DPA merit a systematic examination of the elongase enzymes involved in their metabolism. Human and rat elongase-5 (Elovl5) elongate C18 and C20 PUFA [4,5] and human and mouse elongase-2 (Elovl2) are active with C20 and C22 PUFA [6]. However, the putative substrate preferences for these enzymes have been inferred from separate studies, each using single concentrations of substrate. Accordingly, these studies provide little insight into the reasons for EPA and/or DPA accumulation when upstream precursor fatty acids are abundant. Beyond DPA formation, Fads2 may have an additional influence on the pathway. In addition to metabolising ALA, it also metabolises 24:5n-3, a progenitor of DHA. Since Fads2 is used by two substrates in the pathway from ALA to DHA synthesis, there is intrinsic potential for competitive substrate inhibition. Substrate inhibition has been suggested as an explanation for increasing EPA and DPA, but decreasing DHA when dietary ALA is abundant [7,8]. However, direct competition between ALA and 24:5n-3 with regard to Fads2 has not been reported. We report herein comparative studies of the rat elongases with regard to expression, substrate selectivities, competitive substrate interactions, and dose response curves. We also examined the possible competitive substrate interactions for rat Fads2. The enzyme activity studies used a yeast heterologous expression system. PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29662