Use ofBroken-Symmetry Density Functional Theory ToCharacterize the IspH Oxidized State: Implications for IspH Mechanismand Inhibition

摘要

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 evaluategeometries, energies, and Mössbauer signatures of the differentprotonation states that may exist in the oxidized state of the IspHcatalytic cycle. From DFT/SCRF computations performed on the oxidizedstate, we find a state where the substrate, HMBPP, coordinates theapical 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 experimentalgeometry of the active site and that its computed isomer shifts agreewith experiment validate the use of the DFT/SCRF method to assessrelative energies along the IspH reaction pathway. Additional studiesof IspH catalytic intermediates are currently being pursued.