title: Use of Broken-Symmetry Density Functional Theory To Characterize the IspH Oxidized State: Implications for IspH Mechanism and Inhibition.
abstract: With current therapies becoming less efficacious due to increased drug resistance, new inhibitors of both bacterial and malarial targets are desperately needed. The recently discovered methylerythritol phosphate (MEP) pathway for isoprenoid synthesis provides novel targets for the development of such drugs. Particular attention has focused on the IspH protein, the final enzyme in the MEP pathway, which uses its [4Fe-4S] cluster to catalyze the formation of the isoprenoid precursors IPP and DMAPP from HMBPP. IspH catalysis is achieved via a 2e-/2H+reductive dehydroxylation of HMBPP; the mechanism by which catalysis is achieved, however, is highly controversial. The work presented herein provides the first step in assessing different routes to catalysis by using computational methods. By performing broken-symmetry density functional theory (BS-DFT) calculations that employ both the conductor-like screening solvation model (DFT/COSMO) and a finite-difference Poisson-Boltzmann self-consistent reaction field methodology (DFT/SCRF), we evaluate geometries, energies, and Mössbauer signatures of the different protonation states that may exist in the oxidized state of the IspH catalytic cycle. From DFT/SCRF computations performed on the oxidized state, we find a state where the substrate, HMBPP, coordinates the apical iron in the [4Fe-4S] cluster as an alcohol group (ROH) to be one of two, isoenergetic, lowest-energy states. In this state, the HMBPP pyrophosphate moiety and an adjacent glutamate residue (E126) are both fully deprotonated, making the active site highly anionic. Our findings that this low-energy state also matches the experimental geometry of the active site and that its computed isomer shifts agree with experiment validate the use of the DFT/SCRF method to assess relative energies along the IspH reaction pathway. Additional studies of IspH catalytic intermediates are currently being pursued.
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego , 9500 Gilman Drive, Mail Code 0365, La Jolla, California 92093-0365, United States.
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Blachly
authors.affiliation: Department of Chemistry and Biochemistry, Mount Allison University , 63C York Street, Sackville, New Brunswick E4L 1G8, Canada.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Sandala
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital , 262 Danny Thomas Place, Memphis, Tennessee 38105, United States.
authors.forename: Debra Ann
authors.initials: DA
authors.lastname: Giammona
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, Howard Hughes Medical Institute, and Department of Pharmacology, University of California San Diego , La Jolla, California 92093-0365, United States.
authors.forename: Tiqing
authors.initials: T
authors.lastname: Liu
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital , 262 Danny Thomas Place, Memphis, Tennessee 38105, United States.
authors.forename: Donald
authors.initials: D
authors.lastname: Bashford
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego , 9500 Gilman Drive, Mail Code 0365, La Jolla, California 92093-0365, United States ; Skaggs School of Pharmacy and Pharmaceutical Sciences, Howard Hughes Medical Institute, and Department of Pharmacology, University of California San Diego , La Jolla, California 92093-0365, United States ; Skaggs School of Pharmacy and Pharmaceutical Sciences, Howard Hughes Medical Institute, and Department of Pharmacology, University of California San Diego , La Jolla, California 92093-0365, United States.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: McCammon
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute , TPC15, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Louis
authors.initials: L
authors.lastname: Noodleman