Bioorganometallic mechanism of action, and inhibition, of IspH Weixue Wanga, Ke Wangb, Yi-Liang Liua, Joo-Hwan Noa, Jikun Lia, Mark J. Nilgesc, and Eric Oldfielda,b,1 aCenter for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801; bDepartment of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801; cIllinois EPR Research Center, 506 South Mathews Avenue, Urbana, IL 61801 Edited* by Jack Halpern, The University of Chicago, Chicago, IL, and approved December 29, 2009 (received for review September 25, 2009) We have investigated the mechanism of action of Aquifex aeolicus article, we focus on the last enzyme in the nonmevalonate path- IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) re- way, IspH (LytB), with the goal of obtaining a better under- ductase], together with its inhibition, using a combination of standing of its mechanism of action, and inhibition. K V 1 2 13 31 site-directed mutagenesis ( M; max), EPR and H, H, C, P, The IspH (LytB) enzyme HMBPP (E-4-hydroxy-3-methyl- and 57Fe-electron-nuclear double resonance (ENDOR) spectroscopy. but-2-enyl diphosphate) reductase (EC 1.17.1.2) catalyzes the þ − On addition of HMBPP to an (unreactive) E126A IspH mutant, a re- 2H ∕2e reduction of HMBPP (3) to form an approximately action intermediate forms that has a very similar EPR spectrum to 5∶1 mixture of IPP and DMAPP: those seen previously with the HMBPP “parent” molecules, ethy- lene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on 57Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organo- The enzyme is essential for survival and is not found in humans, metallic species with HMBPP, a π∕σ “metallacycle” or η2-alkenyl so is an attractive target for drug development (11). The struc- complex. The complex is poised to interact with Hþ from E126 tures of IspH from Aquifex aeolicus (12) and Escherichia coli (and H124) in reduced wt IspH, resulting in loss of water and for- (13) have recently been reported and indicate trefoil-like protein 1 3 mation of an η -allyl complex. After reduction, this forms an η -allyl structures with a central Fe3S4 cluster (14), whereas EPR (15), π-complex (i.e. containing an allyl anion) that on protonation (at C2 Mössbauer (16, 17), reconstitution and catalytic activity (15, or C4) results in product formation. We find that alkyne dipho- 17) measurements have all been interpreted as indicating that sphates (such as propargyl diphosphate) are potent IspH inhibitors an Fe4S4 cluster is the catalytically active species. Ligand-free and likewise form metallacycle complexes, as evidenced by 1H, 2H, IspH has an “open” structure (12), whereas IspH cocrystallized and 13C ENDOR, where hyperfine couplings of approximately with diphosphate has a “closed” structure (13) in which a serine- 6 MHz for 13C and 10 MHz for 1H, are observed. Overall, the X-asparagine (SXN) loop is involved in hydrogen bonding with a results are of broad general interest because they provide new in- PPi ligand. The mechanism of action of IspH is controversial sights into IspH catalysis and inhibition, involving organometallic and there have been many different proposals (13, 15, 18–21) species, and may be applicable to other Fe4S4-containing proteins, (Fig. S1). However, none of these models has yet been supported such as IspG. by any spectroscopic evidence, and none have led to the devel- opment of IspH inhibitors. Here, we report spectroscopic results enzyme inhibition ∣ iron-sulfur protein ∣ isoprenoid biosynthesis ∣ that indicate the involvement in catalysis of π∕σ metallacycle nonmevalonate pathway intermediates similar to those found for ethylene and allyl alcohol when bound to a nitrogenase FeMo cofactor (22–24). Then, nzymes that catalyze the formation of isoprenoids are of in- based on these results, we show that alkynes can inhibit IspH, Eterest as drug targets. There are two main pathways involved forming once again, metallacyles or π∕σ complexes. in the early steps in isoprenoid biosynthesis: The mevalonate pathway found in animals and in pathogens such as Staphylococ- Results and Discussion cus aureus, Trypanosoma cruzi, and Leishmania spp. (the causative The Role of Protein Residues. We first investigated the role of agents of staph infections, Chagas’ disease and the leishma- protein residues in the IspH mechanism. In previous work, we niases), and the nonmevalonate or Rohmer pathway found in noted that in addition to E126, His42, and His124 were also most pathogenic bacteria, as well as in the malaria parasite, totally conserved residues, were located in the active site region, Plasmodium falciparum (1). Both pathways lead to formation and were likely essential for catalytic activity, a conclusion now of the C5-isoprenoids isopentenyl diphosphate (IPP, 1) and supported by mutagenesis results (13). However, the exact role dimethylallyl diphosphate (DMAPP, 2). In the later stages of iso- of these residues was unclear. We thus determined the KM and prenoid biosynthesis, these C5-compounds then form the farnesyl V max values for three mutants: H42A, H124A, and E126A. In diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) the case of the E126A mutant, activity was so low (V max < 0 025 μ −1 −1 used in protein prenylation, sterol, and carotenoid biosynthesis. mol min mg ) that KM could not be measured. But Understanding how the enzymes catalyzing these “downstream” with the H124A mutant, we found that although V max was low 0 05 μ −1 −1 1 16 μ −1 −1 events function has led to a better understanding of e.g. how FPP ( . mol min mg versus . mol min mg for the μ synthase (2) and GGPP synthase function, and can be inhibited wild-type enzyme), KM was essentially unchanged (7 M versus (3); the discovery that bisphosphonates have potent antiparasitic activity (4); the clinical use of amiodarone (a squalene oxidase Author contributions: W.W., K.W., and E.O. designed research; W.W., K.W., Y.-L.L., J.-H.N., and oxidosqualene cyclase inhibitor) against Chagas’ disease J.L., M.J.N., and E.O. performed research; W.W., J.-H.N., and E.O. analyzed data; K.W. and (5; 6) and leishmaniasis (7); anticancer agents that inhibit both J.L. contributed new reagents/analytic tools; and E.O. wrote the paper. FPPS and GGPPS (8); as well as the discovery that cholesterol The authors declare no conflict of interest. lowering agents (squalene synthase inhibitors) can function as This Direct Submission article had a prearranged editor. antivirulence agents, against S. aureus (9). However, there have 1To whom correspondence may be addressed. E-mail: [email protected]. been few compounds discovered that block the nonmevalonate This article contains supporting information online at www.pnas.org/cgi/content/full/ pathway, fosmidomycin being the notable exception (10). In this 0911087107/DCSupplemental. 4522–4527 ∣ PNAS ∣ March 9, 2010 ∣ vol. 107 ∣ no. 10 www.pnas.org/cgi/doi/10.1073/pnas.0911087107 Downloaded by guest on September 28, 2021 5 μM, for the wild-type enzyme). This indicates that H124 is not a tion) are, interestingly, quite similar to those seen previously with major contributor to substrate binding, but is essential for ethylene and allyl alcohol bound to the α-70Ala mutant of a nitro- catalysis, suggesting that H124 may be involved in delivering Hþ genase FeMo cofactor protein [ethylene: g ¼ 2.123; 1.978; 1.949 to E126 and the bound HMBPP. In the case of H42, however, (24); allyl alcohol: g ¼ 2.123; 1.998; 1.986 (22)] with, on average, we find in the H42A mutant that there is an increase in KM (from only a j0.01j difference between the IspH and nitrogenase g- 7–74 μM), indicating a role in substrate binding, consistent with values. In nitrogenase, ethylene and allyl alcohol have been the crystallographic observation that H42 hydrogen bonds to a shown [via ENDOR and/or density functional theory calculations bound diphosphate ligand (13). There is, nevertheless, also (22–24)] to bind as π∕σ “metallacycles,” as shown e.g. in Fig. 1E. a 5-fold decrease in V max, due perhaps to the possibility that And because HMBPP is simply a substituted alkene, one possible several more distal residues could also be involved in proton explanation of the E126A þ HMBPP spectrum is that HMBPP transfer. binds to reduced IspH in a similar manner, that is, as a π or π∕σ “metallacycle” or “η2-alkenyl” complex, similar to that shown EPR of IspH with Bound Ligands. As shown by Wolff et. al. (15), as- in Fig. 1F (which is based on the nitrogenase/allyl alcohol struc- isolated, oxidized IspH (from E. coli) has low activity and exhibits “ ” þ ture and contains Mo and X ). That such a complex could form an EPR spectrum characteristic of an S ¼ 1∕2 ½Fe3S4 cluster. 3þ 2− with HMBPP is supported by the results of ligand docking calcu- On reconstitution (with DTT, Fe and S ) and under reducing lations using Glide (25) (Fig. 1G and H) in which it can be seen (excess dithionite) conditions, a broad, S ¼ 1∕2 EPR spectrum þ that HMBPP can bind with its diphosphate occupying the “PPi” characteristic of an ½Fe4S4 cluster is obtained (15), essentially site seen crystallographically, at the same time that its double identical to that we find for reduced A. aeolicus IspH (Fig. 1A). bond interacts with the unique fourth Fe, added computationally On addition of HMBPP, the spectrum sharpens and several com- here as described previously (12). Based on the observation that ponents can be seen (Fig.