title: Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity.
abstract: In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo.
authors.affiliation: Institute of Chemical Biology, Imperial College London, Exhibition Road, London SW7 2AZ, UK. r.woscholski@imperial.ac.uk p.gaffney@imperial.ac.uk.
authors.forename: Samuel
authors.initials: S
authors.lastname: Furse
authors.forename: LokHang
authors.initials: L
authors.lastname: Mak
authors.forename: Edward W
authors.initials: EW
authors.lastname: Tate
authors.forename: Richard H
authors.initials: RH
authors.lastname: Templer
authors.forename: Oscar
authors.initials: O
authors.lastname: Ces
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Woscholski
authors.forename: Piers R J
authors.initials: PR
authors.lastname: Gaffney
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D008968:Molecular Conformation; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis* / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D010744:Phosphoric Monoester Hydrolases; C489224:SopB protein, Salmonella