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An ENDOR and HYSCORE investigation of a reaction intermediate in IspG (GcpE) catalysis.

ABSTRACT: IspG is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies on labeled samples, we have specifically assigned the hyperfine interactions in a reaction intermediate. These results help clarify the nature of the reaction intermediate, supporting a direct interaction between the unique fourth Fe in the cluster and C2 and O3 of the ligand.

SUBMITTER: Wang W

PROVIDER: S-EPMC3116442 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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An ENDOR and HYSCORE investigation of a reaction intermediate in IspG (GcpE) catalysis.

Wang Weixue W Wang Ke K Li Jikun J Nellutla Saritha S Smirnova Tatyana I TI Oldfield Eric E

Journal of the American Chemical Society 20110516 22

IspG is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies on labeled samples, we have specifically assigned the hyperfine interactions in a reaction intermediate. These results help clarify the nature of the reaction intermediate, supporting a direct interaction between the unique fourth Fe in the cluster and C2 and O3 of the ligand. ...[more]

PMID: 21574560

Similar Datasets

Inhibition of the 4Fe-4S Proteins IspG and IspH: an EPR, ENDOR and HYSCORE Investigation.

Project description:IspG and IspH are proteins that are involved in isoprenoid biosynthesis in most bacteria as well as in malaria parasites and are important drug targets. They contain cubane-type 4Fe-4S clusters that are involved in unusual 2H+/2e- reductions. Here, we report the results of electron paramagnetic resonance spectroscopic investigations of the binding of amino- and thiolo-HMBPP (HMBPP=E-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate) IspH substrate-analog inhibitors to both proteins, as well as the binding of HMBPP and an acetylene diphosphate inhibitor, to IspG. The results show that amino-HMBPP binds to reduced IspH by Fe-C ?-bonding with the olefinic carbons interacting with the unique 4th Fe in the 4Fe-4S cluster, quite different to the direct Fe-N ligation seen with the oxidized protein. No such ?-complex is observed when amino-HMBPP binds to reduced IspG. No EPR signal is observed with IspH in the presence of dithionite and thiolo-HMBPP, suggesting that the 4Fe-4S cluster is not reduced, consistent with the presence of a 420 nm feature in the absorption spectrum (characteristic of an oxidized cluster). However, with IspG, the EPR spectrum in the presence of dithionite and thiolo-HMBPP is very similar to that seen with HMBPP. The binding of HMBPP to IspG was studied using hyperfine sublevel correlation spectroscopy with 17O and 13C labeled samples: the results rule out direct Fe-O bonding and indicate ?-bonding. Finally, the binding to IspG of a potent acetylene diphosphate inhibitor was studied by using electron-nuclear double resonance spectroscopy with 13C labeled ligands: the large hyperfine couplings indicate strong Fe-C ?-bonding with the acetylenic group. These results illustrate a remarkable diversity in binding behavior for HMBPP-analog inhibitors, opening up new routes to inhibitor design of interest in the context of anti-bacterial and anti-malarial drug discovery, as well as "cubane-type" metallo-biochemistry, in general.

| S-EPMC4078884 | biostudies-literature

Atomic-Resolution Structures of Discrete Stages on the Reaction Coordinate of the [Fe4S4] Enzyme IspG (GcpE).

Project description: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.

| S-EPMC4433817 | biostudies-literature

ENDOR/HYSCORE studies of the common intermediate trapped during nitrogenase reduction of N2H2, CH3N2H, and N2H4 support an alternating reaction pathway for N2 reduction.

Project description:Enzymatic N(2) reduction proceeds along a reaction pathway composed of a sequence of intermediate states generated as a dinitrogen bound to the active-site iron-molybdenum cofactor (FeMo-co) of the nitrogenase MoFe protein undergoes six steps of hydrogenation (e(-)/H(+) delivery). There are two competing proposals for the reaction pathway, and they invoke different intermediates. In the 'Distal' (D) pathway, a single N of N(2) is hydrogenated in three steps until the first NH(3) is liberated, and then the remaining nitrido-N is hydrogenated three more times to yield the second NH(3). In the 'Alternating' (A) pathway, the two N's instead are hydrogenated alternately, with a hydrazine-bound intermediate formed after four steps of hydrogenation and the first NH(3) liberated only during the fifth step. A recent combination of X/Q-band EPR and (15)N, (1,2)H ENDOR measurements suggested that states trapped during turnover of the α-70(Ala)/α-195(Gln) MoFe protein with diazene or hydrazine as substrate correspond to a common intermediate (here denoted I) in which FeMo-co binds a substrate-derived [N(x)H(y)] moiety, and measurements reported here show that turnover with methyldiazene generates the same intermediate. In the present report we describe X/Q-band EPR and (14/15)N, (1,2)H ENDOR/HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N(2)H(2), CH(3)N(2)H, and N(2)H(4) in fact generates a common intermediate, I, and show that the N-N bond of substrate has been cleaved in I. Analysis of this finding leads us to conclude that nitrogenase reduces N(2)H(2), CH(3)N(2)H, and N(2)H(4) via a common A reaction pathway, and that the same is true for N(2) itself, with Fe ion(s) providing the site of reaction.

| S-EPMC3156091 | biostudies-literature

Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (IspG) protein.

Project description:Recombinant Escherichia coli cells engineered for the expression of the xylB gene in conjunction with genes of the nonmevalonate pathway were supplied with (13)C-labeled 1-deoxy-D-xylulose. Cell extracts were analyzed directly by NMR spectroscopy. (13)C-labeled 2C-methyl-D-erythritol 2,4-cyclodiphosphate was detected at high levels in cells expressing xylB, ispC, ispD, ispE, and ispF. The additional expression of the gcpE gene afforded 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as an intermediate of the nonmevalonate pathway. Hypothetical mechanisms involving conserved cysteine residues are proposed for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate catalyzed by the GcpE protein.

| S-EPMC64945 | biostudies-literature

Organometallic mechanism of action and inhibition of the 4Fe-4S isoprenoid biosynthesis protein GcpE (IspG).

Project description:We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe(4)S(4) cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using (2)H, (13)C and (17)O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an eta(2)-alkenyl pi- (or pi/sigma) complex with the 4th Fe in the Fe(4)S(4) cluster, and a similar "metallacycle" also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (K(i) approximately 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a (13)C hyperfine coupling of approximately 7 MHz), as well as literature precedent, we propose that the alkyne forms another pi/sigma metallacycle, an eta(2)-alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role.

| S-EPMC2895101 | biostudies-literature

IspG converts an epoxide substrate analogue to (E)-4-hydroxy-3-methylbut-2-enyl diphosphate: implications for IspG catalysis in isoprenoid biosynthesis.

Project description:IspG is an intriguing enzyme in bacteria, parasite, and plant isoprenoid biosynthesis, and its catalytic mechanism remains elusive. We report here the synthesis of (2R,3R)-4-hydroxy-3-methyl-2,3-epoxybutanyl diphosphate (Epoxy-HMBPP), a proposed intermediate in one of the frequently cited mechanistic models. We have also demonstrated that this epoxide analogue is a catalytically competent IspG substrate. This study represents the first mechanistic study of this important enzyme.

| S-EPMC5726269 | biostudies-literature

Structural analysis of transient reaction intermediate in formic acid dehydrogenation catalysis using two-dimensional IR spectroscopy.

Project description:The molecular structure of a catalytically active key intermediate is determined in solution by employing 2D IR spectroscopy measuring vibrational cross-angles. The formate intermediate (2) in the formic acid dehydrogenation reaction catalyzed by a phosphorus-nitrogen PN3P-Ru catalyst is elucidated. Our spectroscopic studies show that the complex features a formate ion directly attached to the Ru center as a ligand, and a proton added to the imine arm of the dearomatized PN3P* ligand. During the catalytic process, the imine arms are not only reversibly protonated and deprotonated, but also interacting with the protic substrate molecules, effectively serving as the local proton buffer to offer remarkable stability with a turnover number (TON) over one million.

| S-EPMC6298111 | biostudies-literature

Pyridine inhibitor binding to the 4Fe-4S protein A. aeolicus IspH (LytB): a HYSCORE Investigation.

Project description:IspH is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using hyperfine sublevel correlation (HYSCORE) spectroscopy, we show that pyridine inhibitors of IspH directly bind to the unique fourth Fe in the 4Fe-4S cluster, opening up new routes to inhibitor design, of interest in the context of both anti-bacterial as well as anti-malarial drug discovery.

| S-EPMC3092291 | biostudies-literature

An Epoxide Intermediate in Glycosidase Catalysis.

Project description:Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E 3) conformation. Kinetic isotope effects (k cat/K M) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.

| S-EPMC7256955 | biostudies-literature

HYSCORE and DFT Studies of Proton-Coupled Electron Transfer in a Bioinspired Artificial Photosynthetic Reaction Center.

Project description:The photosynthetic water-oxidation reaction is catalyzed by the oxygen-evolving complex in photosystem II (PSII) that comprises the Mn4CaO5 cluster, with participation of the redox-active tyrosine residue (YZ) and a hydrogen-bonded network of amino acids and water molecules. It has been proposed that the strong hydrogen bond between YZ and D1-His190 likely renders YZ kinetically and thermodynamically competent leading to highly efficient water oxidation. However, a detailed understanding of the proton-coupled electron transfer (PCET) at YZ remains elusive owing to the transient nature of its intermediate states involving YZ⋅. Herein, we employ a combination of high-resolution two-dimensional 14N hyperfine sublevel correlation spectroscopy and density functional theory methods to investigate a bioinspired artificial photosynthetic reaction center that mimics the PCET process involving the YZ residue of PSII. Our results underscore the importance of proximal water molecules and charge delocalization on the electronic structure of the artificial reaction center.

| S-EPMC7394912 | biostudies-literature

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