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Publication Abstract from PubMed

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 Delta9-18:0-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 Delta9-acyl-CoA Ole1p from Saccharomyces which requires two catalytically-competent subunits for activity.

Half-of-the-Sites Reactivity of the Castor 9-18:0-ACP Desaturase.,Liu Q, Chai J, Moche M, Guy J, Lindqvist Y, Shanklin J Plant Physiol. 2015 Jul 29. pii: pp.00622.2015. PMID:26224800^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.