Crystal structure of recombinant avian wild type and mutant farnesyl diphosphate synthase and the binding modes of substrates and inhibitor compounds

Abstract

The synthesis of farnesyl diphosphate (FPP), a key intermediate in the isoprenoid biosynthetic pathway required for the synthesis of cholesterol and the formation of prenylated proteins, is catalyzed by the enzyme farnesyl diphosphate synthase (FPS). The crystal structure of wild type avian recombinant FPS, the first three-dimensional structure for any prenyltransferase, determined to 2.6 A resolution, exhibits a novel fold composed entirely of {dollar}\alpha{dollar}-helices joined by connecting loops. Two aspartate-rich sequences that are highly conserved among the isoprenyl diphosphate synthase family of prenyltransferases, are essential for enzymatic activity, and proposed to function in substrate binding, were found on opposite walls of a large central cavity, with the aspartate side-chains approximately 12 A apart and facing each other. An analysis of this X-ray structure coupled with information about conserved amino acids obtained from a sequence alignment of approximately 40 isoprenyl diphosphate synthases that synthesize higher chain length isoprenoid diphosphates suggested that the side chains of residues corresponding to F112 and F113 in the avian enzyme were important for controlling prenyl chain growth. An avian FPS F112A/F113S double mutant was found to synthesize long chain isoprene compounds. An X-ray structure analysis of the F112A/F113S mutant in the apo state and with substrates bound produced strong evidence that these mutations caused the observed change in product specificity by directly altering the specificity of the substrate-product binding pocket in the active site of the enzyme. The apo mutant structure showed the proposed binding pocket was increased in depth by 5.8 A as compared to the wild type. Allylic substrates and product were observed, in the holo structures, bound via their pyrophosphate groups to the aspartates of the first aspartate-rich sequence (D117-D121), with the hydrocarbon tails of all the ligands oriented in the same configuration, growing down the hydrophobic pocket toward the mutation site. A model constructed to show how the growth of long chain prenyl product in FPS may proceed by creation of a hydrophobic channel from the FPS active site to the outside surface of the enzyme is presented. Finally, a group of bisphosphonate FPS inhibitors were shown, by x-ray analysis of cocrystal FPS-inhibitor structures, to exert their action by binding to the allylic substrate binding site, and thus blocking the binding of the substrates.