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

Abstract

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 η 2 -alkenyl π-(or π∕σ) 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 ∼ 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a 13 C hyperfine coupling of ∼7 MHz), as well as literature precedent, we propose that the alkyne forms another π∕σ metallacycle, an η 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.4Fe-4S protein | GcpE (IspG) | metallacycle

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

“…Mechanistically then, it appears that MEcPP binds to the conserved Arg/Lys residues in the A domain and that the carbocation that forms on ring opening is located very close to the 4Fe4S cluster domain, which then leads to formation of the reaction intermediate, X (2,16 (18), and an approximately 17 MHz hyperfine coupling for the C2 carbon (16). To see to what extent it might be possible to predict these spectroscopic observables, we used density functional theory (DFT) (19,20).…”

“…It converts 2-C-methyl-D-erythritol-2,4-cyclo diphosphate (MEcPP, 1) to HMBPP (2), which is then converted by a second 4Fe4S protein, IspH [(E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate reductase; EC 1.17.1.2, also known as LytB], to form isopentenyl diphosphate (3) and dimethylallyl diphosphate (4) in an approximately 5∶1 ratio (3). IspG is found in most bacteria as well as in malaria parasites and plants and, because it is essential for pathogen survival and is not produced by humans, is of interest as a drug target.…”

“…drug discovery | metalloprotein | protein folding | iron-sulfur I spG [(E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase; EC1.17.7.1, also known as GcpE] is a 4Fe4S clustercontaining protein (1) involved in isoprenoid biosynthesis in the methylerythritol phosphate pathway (2)(3)(4). It converts 2-C-methyl-D-erythritol-2,4-cyclo diphosphate (MEcPP, 1) to HMBPP (2), which is then converted by a second 4Fe4S protein, IspH [(E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate reductase; EC 1.17.1.2, also known as LytB], to form isopentenyl diphosphate (3) and dimethylallyl diphosphate (4) in an approximately 5∶1 ratio (3).…”

Biochemistry of the non-mevalonate isoprenoid pathway

“…9 Hypothetical reaction mechanisms for IspG protein. a According to [104]; b according to [109]; c according to [115]; d according to [111] Biochemistry of the non-mevalonate isoprenoid pathway 3805 as a model flavoprotein [102], but only recently it was shown that it is an essential protein serving as an obligatory electron transponder for iron-sulfur proteins of the nonmevalonate pathway in Gram-negative bacteria. Plants and apicomplexan parasites are believed to use ferredoxin as electron transponders for IspG and IspH.…”

“…It is generally assumed that the reaction involves a free radical intermediate that is obtained by electron transfer from the iron-sulfur cluster [100,104,105,[109][110][111]. An oxiran intermediate (17) has been proposed to serve as a reaction intermediate [109,[112][113][114][115]. The sequence of electron transfer and bond-breaking steps is still in dispute.…”