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Mechanism of Action and Inhibition of Dehydrosqualene Synthase

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Mechanism of Action and Inhibition of Dehydrosqualene Synthase Mechanism of action and inhibition of dehydrosqualene synthase Fu-Yang Lina,1, Chia-I Liub,c,d,1, Yi-Liang Liua, Yonghui Zhange, Ke Wange, Wen-Yih Jengc,d, Tzu-Ping Koc,d, Rong Caoe, Andrew H.-J. Wangb,c,d,2, and Eric Oldfielda,e,2 aCenter for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; bInstitute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan; cInstitute of Biological Chemistry and dCore Facility for Protein Production and X-ray Structural Analysis, Academia Sinica, Taipei 115, Taiwan; and eDepartment of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Edited* by Frances H. Arnold, California Institute of Technology, Pasadena, CA, and approved October 11, 2010 (received for review July 26, 2010) “Head-to-head” terpene synthases catalyze the first committed converted by the same enzyme to dehydrosqualene, which is steps in sterol and carotenoid biosynthesis: the condensation of further transformed to STX (8). Because STX is a so-called two isoprenoid diphosphates to form cyclopropylcarbinyl diphos- virulence factor for S. aureus (9), inhibiting its formation is of phates, followed by ring opening. Here, we report the structures of interest in the context of developing new routes to antiinfective Staphylococcus aureus dehydrosqualene synthase (CrtM) com- therapies (10). plexed with its reaction intermediate, presqualene diphosphate Given the key role of the head-to-head tri- and tetraterpene (PSPP), the dehydrosqualene (DHS) product, as well as a series of synthases in sterol and carotenoid biosynthesis, there has been inhibitors. The results indicate that, on initial diphosphate loss, the remarkably little work reported on their three-dimensional struc- primary carbocation so formed bends down into the interior of the tures. There has been one report of the structure of human SQS protein to react with C2,3 double bond in the prenyl acceptor to with a bound inhibitor (11), but relatively little mechanistic infor- form PSPP, with the lower two-thirds of both PSPP chains occupy- mation was obtained because the inhibitor was not obviously sub- ing essentially the same positions as found in the two farnesyl strate, intermediate, or product-like. In our group, we reported chains in the substrates. The second-half reaction is then initiated the X-ray crystallographic structure of CrtM from S. aureus (10). by the PSPP diphosphate returning back to the Mg2þ cluster for There were two substrate-analog inhibitor binding sites (sites ionization, with the resultant DHS so formed being trapped in a S1 and S2), but determining which represented the prenyl donor surface pocket. This mechanism is supported by the observation (the “allylic” FPP that ionizes to form the 1′ carbocation) and that cationic inhibitors (of interest as antiinfectives) bind with which represented the prenyl acceptor (that provides the C2,3 their positive charge located in the same region as the cyclopropyl alkene group) was not attempted because the 1′-2,3 distances for carbinyl group; that S-thiolo-diphosphates only inhibit when in both possible assignments were ∼5 Å. In this paper, we provide the allylic site; activity results on 11 mutants show that both structural and mechanistic models for head-to-head terpene DXXXD conserved domains are essential for PSPP ionization; and synthase activity, and inhibition, based on nine X-ray structures; the observation that head-to-tail isoprenoid synthases as well as studies of site-directed mutagenesis based on both structure and CHEMISTRY terpene cyclases have ionization and alkene-donor sites which bioinformatics; chemical reactivity; and comparisons with head- spatially overlap those found in CrtM. to-head terpene synthases and terpene cyclases. triterpene ∣ X-ray crystallography ∣ drug discovery ∣ staphyloxanthin ∣ Results and Discussion quinuclidine Structure of PSPP Bound to CrtM. The overall reactions catalyzed by CrtM, SQS, and PSYare shown in Fig. 1 and involve ionization of ead-to-head terpene synthases catalyze the first committed one isoprenoid diphosphate molecule to form a 1′ carbocation, Hsteps in the biosynthesis of sterols and carotenoid pigments: which then undergoes nucleophilic attack by the C2,3 double bond the C1′-2,3 condensation of two isoprenoid diphosphates to form in the second isoprenoid diphosphate to form, after proton loss, a cyclopropylcarbinyl diphosphate (1, 2), followed by ring open- PSPP or PPPP. After a second ionization, these molecules then ing to form squalene, dehydrosqualene, or phytoene. In humans undergo a complex series of rearrangements (12) thought to in- and in many pathogenic yeasts, fungi, and protozoa, as well as in volve cyclopropyl and cyclobutyl species to form either primarily plants, the isoprenoid diphosphate is farnesyl diphosphate (FPP) dehydrosqualene (with CrtM; or SQS in the absence of NADPH); and the initial product is the C30 isoprenoid, presqualene diphos- squalene (with SQS, in the presence of NADPH), or phytoene phate (PSPP). As implied by its name, PSPP is then converted (by (with PSY). But which site houses the cation (donor), and which the same enzyme as used in the condensation reaction, squalene synthase, SQS) to squalene which, after epoxidation, is cyclized to lanosterol (3), as shown in Fig. 1. Lanosterol then undergoes Author contributions: F.-Y.L., C.-I.L., Y.-L.L., R.C., A.H.-J.W., and E.O. designed research; F.-Y.L., C.-I.L., Y.-L.L., Y.Z., K.W., W.-Y.J., T.-P.K., R.C., and E.O. performed research; F.-Y.L., numerous additional reactions, resulting in formation of sterols, C.-I.L., Y.-L.L., W.-Y.J., T.-P.K., R.C., and E.O. analyzed data; Y.Z. and K.W. contributed key cell membrane components. As such, squalene synthase new reagents/analytic tools; and A.H.-J.W. and E.O. wrote the paper. inhibitors are of interest as antiparasitics, in particular against The authors declare no conflict of interest. Trypanosoma cruzi (4) and Leishmania spp. (5), the causative *This Direct Submission article had a prearranged editor. agents of Chagas disease and the leishmaniases. In plants, the Data deposition: The atomic coordinates and structure factors have been deposited in the related enzyme phytoene synthase (PSY) catalyzes the condensa- Research Collaboratory for Structural Bioinformatics Protein Data Bank, www.pdb.org for tion of two C20 isoprenoid diphosphate (geranylgeranyl diphos- dehydrosqualene synthase complexed with presqualene diphosphate (PDB ID codes 3LGZ, phate, GGPP) molecules (6) to form prephytoene diphosphate 3ADZ, 3NPR), dehydrosqualene (PDB ID code 3NRI), BPH-651 (PDB ID code 3ACW), BPH-673 (PDB ID code 3ACX), BPH-702 (PDB ID code 3ACY), and GGSPP (PDB ID code 3AE0), and for (PPPP) that, after ring opening, forms phytoene, which is then human squalene synthase complexed with BPH-652 (PDB ID code 3LEE). converted to carotenoid pigments (7) (Fig. 1). In the bacterium 1F-Y.L. and C-I.L. contributed equally to this work. Staphylococcus aureus , the initial step in formation of the caro- 2To whom correspondence may be addressed. E-mail: [email protected] or ahjwang@ tenoid pigment staphyloxanthin (STX) is carried out by conden- gate.sinica.edu.tw. sation of two FPP molecules by the enzyme dehydrosqualene This article contains supporting information online at www.pnas.org/lookup/suppl/ synthase (CrtM), again initially forming PSPP (8), which is then doi:10.1073/pnas.1010907107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1010907107 PNAS ∣ December 14, 2010 ∣ vol. 107 ∣ no. 50 ∣ 21337–21342 Fig. 1. Schematic illustration of the reactions catalyzed by head-to-head terpene synthases: CrtM, SQS, and PSY. All reactions involve an initial C1′-2,3 cyclo- propanation step. The end products of the biosynthetic pathways are highly varied and include sterols (cholesterol, ergosterol) and carotenoids (staphylox- anthin, β-carotene). is the alkene (acceptor)? How do PSPP and dehydrosqualene protein, which is known to be unreactive (13). The Y129A CrtM (DHS) bind to CrtM (or SQS)? And where do the diphosphates mutant was indeed unreactive, with either FPP (0% activity) or ionize? In our work on CrtM (10), we used the nonreactive PSPP (7% activity, Fig. S1) as substrate, indicating that both the substrate analog, S-thiolo-farnesyl diphosphate (FSPP), to define first-half (PSPP formation) as well as the second-half (PSPP to two prenyl diphosphate binding sites: S1 and S2. The structure dehydrosqualene) reactions were inhibited, which enabled us to obtained (PDB ID code 2ZCP) is shown in Fig. 2A from which obtain diffraction quality crystals of a CrtM/PSPP complex. Our it can be seen that the C1′-C2,3 distances for the two possible initial CrtM/PSPP crystals diffracted to 2.4 Å (full crystallographic mechanistic models (i.e., S1 ¼ donor or S1 ¼ acceptor) are very data and structure refinement details are given in Table S1), and similar (5–5.4 Å) making it impossible to make reliable donor– using the CrtM-FSPP structure (PDB ID code 2ZCP) minus acceptor site assignments based solely on this metric. ligands as a template, we obtained the electron density results We therefore produced a CrtM mutant (Y129A) that, based on shown in Fig. S2A (PDB ID code 3LGZ). We then obtained a sec- sequence alignment with rat SQS, corresponds to Y171 in the rat ond structure (electron density shown in Fig. 2B) with improved Fig. 2. Crystallographic results for CrtM with a bound substrate-like inhibitor FSPP and the intermediate PSPP. (A) Bound FSPP (PDB code ID 2ZCP), from ref. 10. (B) The 2Fo-Fc map for PSPP contoured at 1σ (green) and 3σ (red) (PDB code ID 3ADZ). (C) Superposition of PSPP (PDB ID code 3ADZ) on FSPP in CrtM. (D) Close- up view of PSPP-FSPP superposition. Arrows indicate movements of the C1′, C2, and C3 atoms. 21338 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1010907107 Lin et al.
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