title: Atomic-Resolution Structures of Discrete Stages on the Reaction Coordinate of the [Fe4S4] Enzyme IspG (GcpE).
abstract: IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent and an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG-catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate into (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate. In addition, the enzyme's structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism, which describes all stages from initial ring opening to formation of (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many prokaryotic and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence.
authors: Center for Integrated Protein Science, Department Chemie, Lehrstuhl für Biochemie, Technische Universität München, Garching D-85747, Germany.
authors: Felix
authors: F
authors: Quitterer
authors: Center for Integrated Protein Science, Department Chemie, Lehrstuhl für Biochemie, Technische Universität München, Garching D-85747, Germany.
authors: Annika
authors: A
authors: Frank
authors: Department of Chemistry, University of Illinois, Urbana, IL 61801, USA.
authors: Ke
authors: K
authors: Wang
authors: Department of Chemistry, University of Illinois, Urbana, IL 61801, USA.
authors: Guodong
authors: G
authors: Rao
authors: Department of Chemistry, University of Illinois, Urbana, IL 61801, USA.
authors: Bing
authors: B
authors: O'Dowd
authors: Department of Chemistry, University of Illinois, Urbana, IL 61801, USA.
authors: Jikun
authors: J
authors: Li
authors: Department of Chemistry, University of Illinois, Urbana, IL 61801, USA.
authors: Francisco
authors: F
authors: Guerra
authors: Division of Physical Sciences and Engineering, KAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal 23955, Kingdom of Saudi Arabia.
authors: Safwat
authors: S
authors: Abdel-Azeim
authors: Center for Integrated Protein Science, Department Chemie, Lehrstuhl für Biochemie, Technische Universität München, Garching D-85747, Germany.
authors: Adelbert
authors: A
authors: Bacher
authors: Division of Physical Sciences and Engineering, KAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal 23955, Kingdom of Saudi Arabia.
authors: Jörg
authors: J
authors: Eppinger
authors: Department of Chemistry, University of Illinois, Urbana, IL 61801, USA.
authors: Eric
authors: E
authors: Oldfield
authors: Center for Integrated Protein Science, Department Chemie, Lehrstuhl für Biochemie, Technische Universität München, Garching D-85747, Germany.
authors: Michael
authors: M
authors: Groll
mesh_terms: D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D004798:Enzymes; D011994:Recombinant Proteins; C421242:hydroxymethylbutenyl 4-diphosphate synthase
keywords: iron–sulfur cluster catalysis; methylerythritol-phosphate pathway; reaction mechanisms; terpene biosynthesis