Evidence That the Yeast Desaturase Ole1p Exists as a Dimer in Vivo

Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2010; 285(25):19384-90. DOI: 10.1074/jbc.M110.125377
Source: PubMed


Desaturase enzymes are composed of two classes, the structurally well characterized soluble class found predominantly in the plastids of higher plants and the more widely distributed but poorly structurally defined integral membrane class. Despite their distinct evolutionary origins, the two classes both require an iron cofactor and molecular oxygen for activity and are inhibited by azide and cyanide, suggesting strong mechanistic similarities. The fact that the soluble desaturase is active as a homodimer prompted us test the hypothesis that an archetypal integral membrane desaturase from Saccharomyces cerevisiae, the Delta(9)-acyl-Co-A desaturase Ole1p, also exhibits a dimeric organization. Ole1p was chosen because it is one of the best characterized integral membrane desaturase and because it retains activity when fused with epitope tags. FLAG-Ole1p was detected by Western blotting of immunoprecipitates in which anti-Myc antibodies were used for capture from yeast extracts co-expressing Ole1p-Myc and Ole1p-FLAG. Interaction was confirmed by two independent bimolecular complementation assays (i.e. the split ubiquitin system and the split luciferase system). Co-expression of active and inactive Ole1p subunits resulted in an approximately 75% suppression of the accumulation of palmitoleic acid, demonstrating that the physiologically active form of Ole1p in vivo is the dimer in which both protomers must be functional.

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    • "Previously, we demonstrated that the D9-acyl-CoA membrane-bound desaturase Ole1p from bakers' yeast and a series of higher plant membrane desaturases, fatty acid desaturase2 (FAD2), FAD3, FAD6, FAD7, and FAD8, like the evolutionarily unrelated soluble desaturases , form dimers in vivo (Lou and Shanklin, 2010; Lou et al., 2014). For both the yeast Ole1 (Lou and Shanklin, 2010) and plant FAD2 desaturases (Chapman et al., 2001, 2008), coexpression of inactive mutant subunits results in the inactivation of the endogenous wild-type desaturases, presumably by the formation of heterodimers. Thus, for the membrane class of desaturase enzymes, two catalytically competent subunits are required for catalysis. "
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    ABSTRACT: Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast, animals and bacteria. Both classes exhibit a dimeric quaternary structure. Here we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-ACP desaturase, specifically the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified and its properties validated by X-ray crystallography, size exclusion chromatography and activity assay. Heterodimers comprising distinctly tagged wild type (WT) and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only half the number of active sites, purified heterodimers exhibit equivalent activity to WT homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA Ole1p from Saccharomyces which requires two catalytically-competent subunits for activity. Copyright © 2015, Plant Physiology.
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