Bioorganometallic mechanism of action, and inhibition, of IspH

Abstract

We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M ; V max ), EPR and 1 H, 2 H, 13 C, 31 P, and 57 Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on 57 Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π∕σ "metallacycle" or η 2 -alkenyl complex. The complex is poised to interact with H þ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η 1 -allyl complex. After reduction, this forms an η 3 -allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by 1 H, 2 H, and 13 C ENDOR, where hyperfine couplings of approximately 6 MHz for 13 C and 10 MHz for 1 H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe 4 S 4 -containing proteins, such as IspG.enzyme inhibition | iron-sulfur protein | isoprenoid biosynthesis | nonmevalonate pathway E nzymes that catalyze the formation of isoprenoids are of interest as drug targets. There are two main pathways involved in the early steps in isoprenoid biosynthesis: The mevalonate pathway found in animals and in pathogens such as Staphylococcus aureus, Trypanosoma cruzi, and Leishmania spp. (the causative agents of staph infections, Chagas' disease and the leishmaniases), and the nonmevalonate or Rohmer pathway found in most pathogenic bacteria, as well as in the malaria parasite, Plasmodium falciparum (1). Both pathways lead to formation of the C 5 -isoprenoids isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2). In the later stages of isoprenoid biosynthesis, these C 5 -compounds then form the farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) used in protein prenylation, sterol, and carotenoid biosynthesis. Understanding how the enzymes catalyzing these "downstream" events function has led to a better understanding of e.g. how FPP synthase (2) and GGPP synthase function, and can be inhibited (3); the discovery that bisphosphonates have potent antiparasitic activity (4); the clinical use of amiodarone (a squalene oxidase and oxidosqualene cyclase inhibitor) against Chagas' disease (5; 6) and leishmaniasis (7); anticancer agents that inhibit both FPPS and GGPPS (8); as wel...

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

“…R55, R101, and R128 have been shown to be essential for catalysis in A. aeolicus IspG by Lee et al (7) and R141 (Thermus numbering) is essential for survival in Salmonella enterica (17). So, based on the docking results, their conserved nature (Table S1) and the mutagenesis results, these residues are all likely to be involved in MEcPP, reactive intermediate or HMBPP product binding, via their diphosphate groups, while the reactive intermediate and HMBPP product also interact directly with Fe via Fe-O and/or Fe-C bonds, the HMBPP O-1 Fe interaction being similar to that seen with HMBPP binding to IspH (18).…”

“…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).…”

Functional Evidence for the Critical Amino-Terminal Conserved Domain and Key Amino Acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE

“…Studies from Aquifex aeolicus and E. coli indicate that IspH has a trefoil-like structure with a central Fe 4 S 4 cluster, which is coordinated by the Cys residues located in each of the three folding domains (Rekittke et al, 2008;Gräwert et al, 2009). The central cavity is the active site, and the iron-sulfur cluster serves as an electrontransfer cofactor in the active site (Gräwert et al, 2004(Gräwert et al, , 2009(Gräwert et al, , 2010Rekittke et al, 2008;Wang et al, 2010). The IspH mechanism of action is still under debate, but there is considerable evidence in support of the organometallic hypothesis (Wang et al, 2010Span et al, 2012a;Xu et al, 2012;Li et al, 2013).…”

“…The central cavity is the active site, and the iron-sulfur cluster serves as an electrontransfer cofactor in the active site (Gräwert et al, 2004(Gräwert et al, , 2009(Gräwert et al, , 2010Rekittke et al, 2008;Wang et al, 2010). The IspH mechanism of action is still under debate, but there is considerable evidence in support of the organometallic hypothesis (Wang et al, 2010Span et al, 2012a;Xu et al, 2012;Li et al, 2013). The substrate HMBPP initially forms a hydroxy complex with the iron-sulfur cluster, and a subsequent series of complexes with rare organometallic bonds leads to the synthesis of IPP and DMAPP (Wang et al, 2010Li et al, 2013).…”

“…The IspH mechanism of action is still under debate, but there is considerable evidence in support of the organometallic hypothesis (Wang et al, 2010Span et al, 2012a;Xu et al, 2012;Li et al, 2013). The substrate HMBPP initially forms a hydroxy complex with the iron-sulfur cluster, and a subsequent series of complexes with rare organometallic bonds leads to the synthesis of IPP and DMAPP (Wang et al, 2010Li et al, 2013).…”