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Bioorganometallic chemistry with IspG and IspH: structure, function, and inhibition of the [Fe(4)S(4)] proteins involved in isoprenoid biosynthesis.

ABSTRACT: Enzymes of the methylerythritol phosphate pathway of isoprenoid biosynthesis are attractive anti-infective drug targets. The last two enzymes of this pathway, IspG and IspH, are [Fe4 S4 ] proteins that are not produced by humans and catalyze 2?H(+) /?2 e(-) reductions with novel mechanisms. In this Review, we summarize recent advances in structural, mechanistic, and inhibitory studies of these two enzymes. In particular, mechanistic proposals involving bioorganometallic intermediates are presented, and compared with other mechanistic possibilities. In addition, inhibitors based on substrate analogues as well as developed by rational design and compound-library screening, are discussed. The results presented support bioorganometallic catalytic mechanisms for IspG and IspH, and open up new routes to anti-infective drug design targeting [Fe4 S4 ] clusters in proteins.

SUBMITTER: Wang W

PROVIDER: S-EPMC3997630 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Bioorganometallic chemistry with IspG and IspH: structure, function, and inhibition of the [Fe(4)S(4)] proteins involved in isoprenoid biosynthesis.

Wang Weixue W Oldfield Eric E

Angewandte Chemie (International ed. in English) 20140131 17

Enzymes of the methylerythritol phosphate pathway of isoprenoid biosynthesis are attractive anti-infective drug targets. The last two enzymes of this pathway, IspG and IspH, are [Fe4 S4 ] proteins that are not produced by humans and catalyze 2 H(+) / 2 e(-) reductions with novel mechanisms. In this Review, we summarize recent advances in structural, mechanistic, and inhibitory studies of these two enzymes. In particular, mechanistic proposals involving bioorganometallic intermediates are present ...[more]

PMID: 24481599

Similar Datasets

The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein.

Project description:Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-d-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol x min(-1) x mg(-1). Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 micromol x min(-1) x mg(-1). IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol x min(-1) x mg(-1). The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.

| S-EPMC149876 | biostudies-literature

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

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.

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

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Structure, function and inhibition of the two- and three-domain 4Fe-4S IspG proteins.

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| S-EPMC3365180 | biostudies-literature

Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis.

Project description:X-ray structures of two enzymes in the sterol/isoprenoid biosynthesis pathway have been determined in a structural genomics pilot study. Mevalonate-5-diphosphate decarboxylase (MDD) is a single-domain alpha/beta protein that catalyzes the last of three sequential ATP-dependent reactions which convert mevalonate to isopentenyl diphosphate. Isopentenyl disphosphate isomerase (IDI) is an alpha/beta metalloenzyme that catalyzes interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, which condense in the next step toward synthesis of sterols and a host of natural products. Homology modeling of related proteins and comparisons of the MDD and IDI structures with two other experimentally determined structures have shown that MDD is a member of the GHMP superfamily of small-molecule kinases and IDI is similar to the nudix hydrolases, which act on nucleotide diphosphatecontaining substrates. Structural models were produced for 379 proteins, encompassing a substantial fraction of both protein superfamilies. All three enzymes responsible for synthesis of isopentenyl diphosphate from mevalonate (mevalonate kinase, phosphomevalonate kinase, and MDD) share the same fold, catalyze phosphorylation of chemically similar substrates (MDD decarboxylation involves phosphorylation of mevalonate diphosphate), and seem to have evolved from a common ancestor. These structures and the structural models derived from them provide a framework for interpreting biochemical function and evolutionary relationships.

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IspH-RPS1 and IspH-UbiA: "Rosetta Stone" Proteins.

Project description:The protein IspH, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMPPP) reductase, is an essential 4Fe-4S cluster-containing protein in the methylerythritol phosphate pathway for isoprenoid biosynthesis. Using a sequence similarity network we found that there are >400 IspH proteins that are about twice as large as most of the IspHs studied to date since their IspH domains are fused to either the ribosomal protein S1 (RPS1), or to a UbiA (4-hydroxybenzoate octaprenyltransferase)-like protein. Many of the IspH-RPS1 proteins are present in anaerobes found in the human gut and some, such as Clostridium botulinum, C. tetani and Fusobacterium nucleatum, are pathogens. The IspH-UbiAs are all found in sulfate-reducing anaerobes. The IspH domains in IspH-RPS1 are fused to 4 and in a few cases 6 tandem repeats in RPS1 that, in most organisms, bind to mRNA or form part of the bacterial ribosome. Mutants in which the four RPS1 domains were sequentially eliminated had similar IspH activity as wild-type protein, indicating they are not essential for IspH catalysis. Overall, the results are of interest since they represent the first isolation of a catalytically active IspH-RPS1, as well as the identification of IspH-UbiA hybrids, two "Rosetta stone" proteins that are likely to be functionally related-IspH producing the isoprenoids required for a UbiA-like prenyl transferase; the IspH-RPS1 hybrids, perhaps, being involved in the stringent response or as Fe/O2 sensors.

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Inhibition Studies on Enzymes Involved in Isoprenoid Biosynthesis: Focus on Two Potential Drug Targets: DXR and IDI-2 Enzymes.

Project description:Isoprenoid compounds constitute an immensely diverse group of acyclic, monocyclic and polycyclic compounds that play important roles in all living organisms. Despite the diversity of their structures, this plethora of natural products arises from only two 5-carbon precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This review will discuss the enzymes in the mevalonate (MVA) and methylerythritol phosphate (MEP) biosynthetic pathways leading to IPP and DMAPP with a particular focus on MEP synthase (DXR) and IPP isomerase (IDI), which are potential targets for the development of antibiotic compounds. DXR is the second enzyme in the MEP pathway and the only one for which inhibitors with antimicrobial activity at pharmaceutically relevant concentrations are known. All of the published DXR inhibitors are fosmidomycin analogues, except for a few bisphosphonates with moderate inhibitory activity. These far, there are no other candidates that target DXR. IDI was first identified and characterised over 40 years ago (IDI-1) and a second convergently evolved isoform (IDI-2) was discovered in 2001. IDI-1 is a metalloprotein found in Eukarya and many species of Bacteria. Its mechanism has been extensively studied. In contrast, IDI-2 requires reduced flavin mononucleotide as a cofactor. The mechanism of action for IDI-2 is less well defined. This review will describe how lead inhibitors are being improved by structure-based drug design and enzymatic assays against DXR to lead to new drug families and how mechanistic probes are being used to address questions about the mechanisms of the isomerases.

| S-EPMC3856697 | biostudies-literature

Isoprenoid Biosynthesis in Pathogenic Bacteria: Nuclear Resonance Vibrational Spectroscopy Provides Insight into the Unusual [4Fe-4S] Cluster of the E.?coli LytB/IspH Protein.

Project description:The LytB/IspH protein catalyzes the last step of the methylerythritol phosphate (MEP) pathway which is used for the biosynthesis of essential terpenoids in most pathogenic bacteria. Therefore, the MEP pathway is a target for the development of new antimicrobial agents as it is essential for microorganisms, yet absent in humans. Substrate-free LytB has a special [4Fe-4S](2+) cluster with a yet unsolved structure. This motivated us to use synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) in combination with quantum chemical-molecular mechanical (QM/MM) calculations to gain more insight into the structure of substrate-free LytB. The apical iron atom of the [4Fe-4S](2+) is clearly linked to three water molecules. We additionally present NRVS data of LytB bound to its natural substrate, (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) and to the inhibitors (E)-4-amino-3-methylbut-2-en-1-yl diphosphate and (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate.

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