title: X-ray crystallographic studies of substrate binding to aristolochene synthase suggest a metal ion binding sequence for catalysis.
abstract: The universal sesquiterpene precursor, farnesyl diphosphate (FPP), is cyclized in an Mg(2+)-dependent reaction catalyzed by the tetrameric aristolochene synthase from Aspergillus terreus to form the bicyclic hydrocarbon aristolochene and a pyrophosphate anion (PP(i)) coproduct. The 2.1-A resolution crystal structure determined from crystals soaked with FPP reveals the binding of intact FPP to monomers A-C, and the binding of PP(i) and Mg(2+)(B) to monomer D. The 1.89-A resolution structure of the complex with 2-fluorofarnesyl diphosphate (2F-FPP) reveals 2F-FPP binding to all subunits of the tetramer, with Mg(2+)(B)accompanying the binding of this analogue only in monomer D. All monomers adopt open activesite conformations in these complexes, but slight structural changes in monomers C and D of each complex reflect the very initial stages of a conformational transition to the closed state. Finally, the 2.4-A resolution structure of the complex with 12,13-difluorofarnesyl diphosphate (DF-FPP) reveals the binding of intact DF-FPP to monomers A-C in the open conformation and the binding of PP(i), Mg(2+)(B), and Mg(2+)(C) to monomer D in a predominantly closed conformation. Taken together, these structures provide 12 independent "snapshots" of substrate or product complexes that suggest a possible sequence for metal ion binding and conformational changes required for catalysis.
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
authors.forename: Ekaterina Y
authors.initials: EY
authors.lastname: Shishova
authors.forename: Fanglei
authors.initials: F
authors.lastname: Yu
authors.forename: David J
authors.initials: DJ
authors.lastname: Miller
authors.forename: Juan A
authors.initials: JA
authors.lastname: Faraldos
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Zhao
authors.forename: Robert M
authors.initials: RM
authors.lastname: Coates
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
authors.forename: David W
authors.initials: DW
authors.lastname: Christianson
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D002384:Catalysis; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism
chemical_list: C525090:12,13-difluorofarnesyl diphosphate; D005656:Fungal Proteins; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D007535:Isomerases; C060050:aristolochene synthase; D008274:Magnesium