Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat 6-desaturase activity

The 6-desaturase catalyzes key steps in longchain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat 6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of 6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and 6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for 6-desaturase activity. —Guillou, H., S. D’Andrea, V. Rioux, R. Barnouin, S. Dalaine, F. Pedrono, S. Jan, and P. Legrand. Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat 6-desaturase activity. J. Lipid Res. 2004. 45: 32–40. Supplementary key words FADS2 • polyunsaturated fatty acid biosynthesis • hexadecenoic acid Long-chain polyunsaturated fatty acids (PUFAs) such as arachidonic acid (C20:4n-6) and docosahexaenoic acid (C22:6n-3) play pivotal roles in a variety of biological functions (1). In animals, some of the daily needs in longchain PUFAs are fulfilled from the diet. However, most of the long-chain PUFAs found in animal tissues are derived from the biosynthetic pathway involving elongations, 6desaturation, and 5-desaturation for conversion of essential fatty acid precursors (C18:2n-6 and C18:3n-3) to their respective 20and 22-carbon polyenoic products. None of the desaturases involved in this biosynthetic pathway have been reproducibly purified, and their structure remains to be characterized. The only animal desaturase whose structure is known is the 9-desaturase (2). This enzyme is part of a multienzyme system present in the endoplasmic reticulum and is composed of 9-desaturase, NADH cytochrome b5 reductase, and cytochrome b5. In the process of double bond formation, the membrane-bound cytochrome b5 transfers electrons by lateral diffusion from NADH cytochrome b5 reductase to the 9 fatty acid desaturase (2). Although the first mammalian 9-desaturase was cloned almost 20 years ago (3), mammalian desaturases involved in PUFA biosynthetic pathways, i.e., 6and 5-desaturases, have been cloned more recently (4–8). Comparison of their respective amino acid sequences shows one major difference between 9-desaturase and 6and 5-desaturases: an N-terminal cytochrome b5-like domain is present in 6and 5-desaturases but not in 9-desaturase. Numerous cytochrome b5-like domains have been identified in various desaturases from yeast, plants, and animals (9). This remarkable characteristic raises the possibility that NADH cytochrome b5 reductase transfers electrons to the catalytic site of these cytochrome b5 fusion desaturases directly via the cytochrome b5-like domain and does not require an independent cytochrome b5. The presence of such cytochrome b5-like domains in desaturase proteins is likely to have originated from a fusion with an ancestral cytochrome b5 gene that may have conferred some evolutionarily selectable advantage. Although these cytochrome Abbreviations: FCS, fetal calf serum; GC, gas chromatography. 1 To whom correspondence should be addressed. e-mail: [email protected] The online version of this article (available at http://www.jlr.org) contains an additional three figures. Manuscript received 1 August 2003 and in revised form 1 October 2003. Published, JLR Papers in Press, October 16, 2003. DOI 10.1194/jlr.M300339-JLR200 by gest, on O cber 3, 2017 w w w .j.org D ow nladed fom 0.DC1.html http://www.jlr.org/content/suppl/2004/01/23/M300339-JLR20 Supplemental Material can be found at: