title: Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase.
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 (Saccharomyces cerevisiae), 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-acyl carrier protein 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 and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type 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 yeast, which requires two catalytically competent subunits for activity.
authors.affiliation: Biological, Environmental and Climate Science, Brookhaven National Laboratory, Upton, New York 11973 (Q.L., J.C., J.S.); andDepartment of Medical Biochemistry and Biophysics, Molecular Structural Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden (M.M., J.G., Y.L.).
authors.forename: Qin
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Biological, Environmental and Climate Science, Brookhaven National Laboratory, Upton, New York 11973 (Q.L., J.C., J.S.); andDepartment of Medical Biochemistry and Biophysics, Molecular Structural Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden (M.M., J.G., Y.L.).
authors.forename: Jin
authors.initials: J
authors.lastname: Chai
authors.affiliation: Biological, Environmental and Climate Science, Brookhaven National Laboratory, Upton, New York 11973 (Q.L., J.C., J.S.); andDepartment of Medical Biochemistry and Biophysics, Molecular Structural Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden (M.M., J.G., Y.L.).
authors.forename: Martin
authors.initials: M
authors.lastname: Moche
authors.affiliation: Biological, Environmental and Climate Science, Brookhaven National Laboratory, Upton, New York 11973 (Q.L., J.C., J.S.); andDepartment of Medical Biochemistry and Biophysics, Molecular Structural Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden (M.M., J.G., Y.L.).
authors.forename: Jodie
authors.initials: J
authors.lastname: Guy
authors.affiliation: Biological, Environmental and Climate Science, Brookhaven National Laboratory, Upton, New York 11973 (Q.L., J.C., J.S.); andDepartment of Medical Biochemistry and Biophysics, Molecular Structural Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden (M.M., J.G., Y.L.).
authors.forename: Ylva
authors.initials: Y
authors.lastname: Lindqvist
authors.affiliation: Biological, Environmental and Climate Science, Brookhaven National Laboratory, Upton, New York 11973 (Q.L., J.C., J.S.); andDepartment of Medical Biochemistry and Biophysics, Molecular Structural Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden (M.M., J.G., Y.L.) shanklin@bnl.gov.
authors.forename: John
authors.initials: J
authors.lastname: Shanklin
mesh_terms: D000213:Acyl Carrier Protein / Q000378:metabolism*; D002367:Ricinus communis / Q000201:enzymology*; D044943:Fatty Acid Desaturases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D000213:Acyl Carrier Protein; D005456:Fluorescent Dyes; D050505:Mutant Proteins; D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases