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The Non-mevalonate Pathway of Isoprenoid Precursor Biosynthesis

2007; Elsevier BV; Volume: 282; Issue: 30 Linguagem: Inglês

10.1074/jbc.r700005200

1083-351X

William N. Hunter,

Pharmacological Effects of Natural Compounds

The recently discovered non-mevalonate biosynthetic route to isoprenoid precursors is an essential metabolic pathway in plants, apicomplexan parasites, and many species of bacteria. The pathway relies on eight enzymes exploiting different cofactors and metal ions. Structural and mechanistic data now exist for most components of the pathway though there remain some gaps in our knowledge. The individual enzymes represent new, validated targets for broad spectrum antimicrobial drug and herbicide development. Detailed knowledge of the pathway may also be exploited to genetically modify microorganisms and plants to produce compounds of agricultural and medical interest. The recently discovered non-mevalonate biosynthetic route to isoprenoid precursors is an essential metabolic pathway in plants, apicomplexan parasites, and many species of bacteria. The pathway relies on eight enzymes exploiting different cofactors and metal ions. Structural and mechanistic data now exist for most components of the pathway though there remain some gaps in our knowledge. The individual enzymes represent new, validated targets for broad spectrum antimicrobial drug and herbicide development. Detailed knowledge of the pathway may also be exploited to genetically modify microorganisms and plants to produce compounds of agricultural and medical interest. Isopentenyl pyrophosphate (IPP) 2The abbreviations used are: IPP, isopentenyl diphosphate; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; CDP-ME, 4-diphosphocytidyl-2C-methyl-d-erythritol; CDP-ME2P, 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate; DMAPP, dimethylallyl diphosphate; DOXP, 1-deoxy-d-xylulose 5-phosphate; DXS, 1-deoxy-d-xylulose-5-phosphate synthase; GHMP, galactose kinase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; IDI, isopentenyl-diphosphate isomerase; IspC, 1-deoxy-d-xylulose-5-phosphate reductoisomerase; MECP, 2C-methyl-d-erythritol-2,4-cyclodiphosphate; MEP, 2C-methyl-d-erythritol-4-phosphate; MVA, mevalonate. Proteins are designated as DXS, IspC-D-E-F-G-H, IDI-I, and IDI-II as defined in the text; genes are abbreviated as dxs, ispC, etc., and species-specific examples given in a genus-species abbreviation, i.e. EcDXS for E. coli DXS. and dimethylallyl pyrophosphate (DMAPP) are the universal precursors of natural products called isoprenoids. This large family, in excess of 35,000 distinct compounds, includes molecules such as sterols, dolichols, triterpenes, ubiquinone, components of macromolecules such as prenyl groups, and isopentenylated tRNAs (1Rohmer M. Barton D. Nakanishi K. Comprehensive Natural Products Chemistry. Vol. 2. Elsevier Science Publishers B.V., Amsterdam1999: 45-67Crossref Google Scholar, 2Sacchettini J.C. Poulter C.D. Science. 1997; 277: 1788-1789Crossref PubMed Scopus (463) Google Scholar, 3Kuzuyama T. Seto H. Nat. Prod. Rep. 2003; 20: 171-183Crossref PubMed Scopus (254) Google Scholar). The diverse chemical properties of isoprenoids are exploited in varied and important biological functions including electron transport, hormone-based signaling, the regulation of transcription and post-translational processes, meiosis, apoptosis, glycoprotein biosynthesis, and protein degradation. In addition, isoprenoids are structural components of cell and organelle membranes. Two biosynthetic routes to IPP and DMAPP have evolved. For many years it was assumed that the mevalonate (MVA) pathway was the sole route to IPP and DMAPP. This pathway uses seven enzymes to supply the precursors in most eukaryotes (all mammals), archaea, a few eubacteria, the cytosol and mitochondria of plants, fungi, and Trypanosoma and Leishmania (4Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4566) Google Scholar). This pathway starts with production of acetoacetyl-CoA from two molecules of acetyl-CoA in a reaction catalyzed by thiolase. A third acetyl-CoA is then condensed with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase. The NADPH-dependent HMG-CoA reductase then converts the CoA derivative to (R)-MVA. Next, in ATP-dependent steps, (R)-MVA is phosphorylated to (R)-MVA 5-diphosphate sequentially by mevalonate kinase and diphosphomevalonate kinase. The diphosphate is subsequently decarboxylated by mevalonate diphosphate decarboxylase to yield a pool of IPP. An IPP isomerase then produces DMAPP from some of the IPP. A wealth of data are available on the constituents of the MVA pathway in large part because of its relevance for human health. The MVA pathway leads to the biosynthesis of cholesterol, and inhibition of HMG-CoA reductase by statins controls production of the sterol with benefits for lowering blood pressure, the treatment of cardiovascular disease, and inflammatory processes (5Liao J.K. Laufs U. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 89-118Crossref PubMed Scopus (1399) Google Scholar). The mevalonate-independent pathway was discovered only recently (6Eisenreich W. Bacher A. Arigoni D. Rohdich F. Cell Mol. Life Sci. 2004; 61: 1401-1426Crossref PubMed Scopus (528) Google Scholar, 7Rodriguez-Concepcion M. Boronat A. Plant Physiol. 2002; 130: 1079-1089Crossref PubMed Scopus (655) Google Scholar, 8Rohmer M. Nat. Prod. Rep. 1999; 16: 565-574Crossref PubMed Scopus (926) Google Scholar). This pathway is called the non-mevalonate route or alternatively, the 1-deoxy-d-xylulose-5-phosphate (DOXP) or 2C-methyl-d-erythritol-4-phosphate (MEP) pathway. This pathway occurs in plant chloroplasts, algae, cyanobacteria, eubacteria, and apicomplexan parasites. Characterization of the DOXP pathway is one of the best examples of modern proteomics. Researchers have exploited NMR methodology to track substrates and products, enzyme-assisted synthesis to acquire reagents necessary to characterize pathway components, and crystallography to provide structural detail that complements enzymatic studies. The DOXP pathway consists of eight reactions catalyzed by nine enzymes, seven of which are characterized structurally (Figs. 1 and 2). Each step will be detailed with a description of the enzyme structure and reactivity. A major impetus to study the pathway is due to the opportunities it offers in biomedicine and biotechnology. These will be explained at the end.FIGURE 2Reactions in stages IV to VIII. Green spheres represent metal ions in IspF and IDI-I. A tetramer of IDI-II subunits is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The non-mevalonate pathway starts with condensation of pyruvate and glyceraldehyde 3-phosphate to produce DOXP, catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase (DXS) using thiamine pyrophosphate as cofactor. DXS has been recalcitrant to crystallographic study. Only recently, assisted by a serendipitous fungal contamination and limited proteolysis, have structures of the Escherichia coli and Deinococcus radiodurans enzymes been reported (9Xiang S. Usunow G. Lange G. Busch M. Tong L. J. Biol. Chem. 2007; 282: 2676-2682Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The DXS subunit is formed by three domains, each similar to equivalent domains in another thiamine-dependent enzyme, transketolase, which catalyzes a similar reaction. However, the arrangement of domains is different in the two enzymes. Like transketolase, the DXS-coenzyme complex is expected to form a covalent adduct between thiamine pyrophosphate and the C2 of pyruvate (10Fiedler E. Thorell S. Sandalova T. Golbik R. Konig S. Schneider G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 591-595Crossref PubMed Scopus (110) Google Scholar). Unlike transketolase where the active site is formed at the dimer interface, the active site of DXS is created with the cofactor buried within a monomer, placing the thiazolium at the base of a polar cavity. The structures required to confirm aspects of specificity for and binding of substrate are lacking. Nevertheless, modeling, and site-directed mutagenesis studies, has taught us about the enzyme activity. This enzyme has been pivotal in the study of this pathway with the discovery that it is a target for the antimicrobial fosmidomycin (11Proteau P.J. Bioorg. Chem. 2004; 32: 483-493Crossref PubMed Scopus (97) Google Scholar, 12Jomaa H. Weisner J. Sanderbrand S. Altinicicek B. Weidemeyer C. Hintz M. Turbachova I. Eberl M. Zeidler J. Lichtenthaler H.K. Soldati D. Beck E. Science. 1999; 285: 1573-1576Crossref PubMed Scopus (1031) Google Scholar). 1-Deoxy-d-xylulose-5-phosphate reductoisomerase (IspC) converts DOXP to MEP. The first structures (of EcIspC) revealed a homodimer with each subunit composed of three domains, an N-terminal cofactor binding domain, a central domain carrying many active site residues, and a flexible segment that acts as a lid for the catalytic center, and a C-terminal helical domain. These structures were incomplete with disorder in and around the active sites (13Yajima S. Nonaka T. Kuzuyama T. Seto H. Ohsawa K. J. Biochem. (Tokyo). 2002; 131: 313-317Crossref PubMed Scopus (95) Google Scholar, 14Reuter K. Sanderbrand S. Jomaa H. Wiesner J. Steinbrecher I. Beck E. Hintz M. Klebe G. Stubbs M.T. J. Biol. Chem. 2002; 277: 5378-5384Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Additional structures with Mn2+, NADPH, fosmidomycin, and other inhibitors have subsequently served to clarify aspects of structure and reactivity (15Steinbacher S. Kaiser J. Eisenreich W. Huber R. Bacher A. Rohdich F. J. Biol. Chem. 2003; 278: 18401-18407Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 16MacSweeney A. Lange R. Fernandes R.P. Schulz H. Dale G.E. Douangamath A. Proteau P.J. Oefner C. J. Mol. Biol. 2005; 345: 115-127Crossref PubMed Scopus (117) Google Scholar, 17Yajima S. Hara K. Sanders J.M. Yin F. Ohsawa K. Wiesner J. Jomaa H. Oldfield E. J. Am. Chem. Soc. 2004; 126: 10824-10825Crossref PubMed Scopus (63) Google Scholar). Structures of IspC from Mycobacterium tuberculosis and Zymomonas mobilis are known (18Ricagno S. Grolle S. Bringer-Meyer S. Sahm H. Lindqvist Y. Schneider G. Biochim. Biophys. Acta. 2004; 1698: 37-44Crossref PubMed Scopus (42) Google Scholar, 19Henriksson L.M. Bjorkelid C. Mowbray S.L. Unge T. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 807-813Crossref PubMed Scopus (34) Google Scholar). IspC is a class B dehydrogenase using the NADPH proS hydride. The mechanism is ordered sequentially with the cofactor binding first. There is a reliance on a divalent cation to polarize and orient the substrate and reaction intermediate. Two mechanisms have been proposed for the initial isomerization that converts DOXP to a methylerythrose intermediate, an α-ketol rearrangement or a retro-adol/aldol reaction (11Proteau P.J. Bioorg. Chem. 2004; 32: 483-493Crossref PubMed Scopus (97) Google Scholar). The intermediate is then reduced by hydride. Fosmidomycin inhibits by coordinating the metal and mimics the business end of DOXP. Here nucleotide derivatives are introduced as substrates and phosphate groups become directly involved in the reactions. MEP reacts with CTP to produce 4-diphosphocytidyl-2C-methyl-d-erythritol (CDP-ME) and release pyrophosphate in the IspD-catalyzed reaction. Of note is the dissection of the EcIspD structure-mechanism relationship by Richard et al. (20Richard S.B. Bowman M.E. Kwiatkowski W. Kang I. Chow C. Lillo A.M. Cane D.E. Noel J.P. Nat. Struct. Biol. 2001; 8: 641-648Crossref PubMed Scopus (104) Google Scholar) based on the combination of informative structures and kinetic analyses (21Richard S.B. Lillo A.M. Tetzlaff C.N. Bowman M.E. Noel J.P. Cane D.E. Biochemistry. 2004; 43: 12189-12197Crossref PubMed Scopus (49) Google Scholar). The enzyme subunit is a single domain α/β structure constructed around a seven-stranded twisted β-sheet into which is inserted an extended "β-arm." Two arms associate to form a dimer interface that involves numerous hydrogen bonds and salt bridges. The active site is created at this interface by seven polypeptide segments, six from one subunit and one from the partner. The study of Arabidopsis thaliana IspD identified alterations in the alignment of subunits with respect to each other (22Gabrielsen M. Kaiser J. Rohdich F. Eisenreich W. Laupitz R. Bacher A. Bond C.S. Hunter W.N. FEBS J. 2006; 273: 1065-1073Crossref PubMed Scopus (28) Google Scholar). An ordered sequential mechanism applies with CTP, probably as an ion pair with Mg2+, binding first. A basic active site binds the four phosphates of the substrates, and in particular lysine residues (Lys-27 and Lys-213 in EcIspD) are key to the stabilization of a pentavalent transition state generated following an in-line nucleophilic attack by the MEP phosphate on the α-phosphate of CTP. Additional structures of Thermatoga maritima, Neisseria gonorrhea IspD, and the Camphylobacter jejuni bifunctional IspDF are now published (23Gabrielsen M. Bond C.S. Hallyburton I. Hecht S. Bacher A. Eisenreich W. Rohdich F. Hunter W.N. J. Biol. Chem. 2004; 279: 52753-52761Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) or in the Protein Data Bank. The ATP-dependent IspE is a member of the GHMP kinase superfamily (24Bork P. Sander C. Valencia A. Protein Sci. 1993; 2: 31-40Crossref PubMed Scopus (348) Google Scholar). The use of a non-hydrolyzable ATP derivative allowed a dead-end complex with EcIspE and CDP-ME to be characterized (25Miallau L. Alphey M.S. Kemp L.E. Leonard G.A. McSweeney S.M. Hecht S. Bacher A. Eisenreich W. Rohdich F. Hunter W.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9173-9178Crossref PubMed Scopus (92) Google Scholar). In addition a structure of Thermus thermophilus IspE is published (26Wada T. Kuzuyama T. Satoh S. Kuramitsu S. Yokoyama S. Unzai S. Tame J.R. Park S.Y. J. Biol. Chem. 2003; 278: 30022-30027Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The enzyme displays the characteristic GHMP kinase α/β fold, arranged into cofactor and substrate-binding domains. The catalytic center is located in a deep cavity near the interface of these domains. Here, in EcIspE, a lysine-aspartate pair (Lys-10 and Asp-141) forms hydrogen bonds with substrate and polarizes the hydroxyl group O2M (Fig. 1) to facilitate proton abstraction with Asp-141 acting as a general base. The basic lysine contributes to stabilization of the transition state. The cofactor γ-phosphate accepts nucleophilic attack from CDP-ME O2M with an associative in-line mechanism to produce 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate (CDP-ME2P) and ADP. In contrast to other GHMP kinases there is no evidence for divalent metal ion binding to the cofactor, and the cofactor purine forms hydrogen-bonding interactions that stabilize the less common syn orientation of the base with respect to the glycosidic bond. Crystal structures of the trimeric IspF from E. coli, Haemophilus influenzae, Shewanella oneidensis, T. thermophilus, and the bifunctional IspDF of C. jejuni are known (23Gabrielsen M. Bond C.S. Hallyburton I. Hecht S. Bacher A. Eisenreich W. Rohdich F. Hunter W.N. J. Biol. Chem. 2004; 279: 52753-52761Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 27Kemp L.E. Bond C.S. Hunter W.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6591-6596Crossref PubMed Scopus (87) Google Scholar, 28Steinbacher S. Kaiser J. Wungsintaweekul J. Hecht S. Eisenreich W. Gerhardt S. Bacher A. Rohdich F. J. Mol. Biol. 2002; 316: 79-88Crossref PubMed Scopus (82) Google Scholar, 29Richard S.B. Ferrer J.L. Bowman M.E. Lillo A.M. Tetzlaff C.N. Cane D.E. Noel J.P. J. Biol. Chem. 2002; 277: 8667-8672Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 30Kishida H. Wada T. Unzai S. Kuzuyama T. Takagi M. Terada T. Shirouzu M. Yokoyama S. Tame J.R. Park S.Y. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 23-31Crossref PubMed Scopus (38) Google Scholar, 31Ni S. Robinson H. Marsing G.C. Bussiere D.E. Kennedy M.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1949-1957Crossref PubMed Scopus (24) Google Scholar). The EcIspF subunit is a small single α/β domain and consists of a four-stranded mixed β-sheet on one side with three α helices on the other. Three subunits form a compact bell-shaped assembly, and a surface area equivalent to that of one subunit is buried on oligomerization. Each active site is built from residues of two adjacent subunits. Here, a Zn2+, with tetrahedral coordination to a pair of histidine residues, an aspartate, and either a water or phosphate if substrate is present, has been identified (27Kemp L.E. Bond C.S. Hunter W.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6591-6596Crossref PubMed Scopus (87) Google Scholar, 28Steinbacher S. Kaiser J. Wungsintaweekul J. Hecht S. Eisenreich W. Gerhardt S. Bacher A. Rohdich F. J. Mol. Biol. 2002; 316: 79-88Crossref PubMed Scopus (82) Google Scholar). Another divalent cation (Mn2+ or under physiological conditions Mg2+) with octahedral coordination is positioned between α- and β-phosphates. The enzyme requires both cations for catalysis, one of which (Zn2+) is always present in the active site and the other presumably brought in with the substrate. Both metal ions contribute to the alignment of the α- and β-phosphates and polarize them. An associative in-line mechanism is likely with, as the first step, nucleophilic attack by the terminal phosphate of CDP-ME2P on the β-phosphate (Fig. 2). This would generate a pentacoordinate transition state, also stabilized by metal ions, which collapses to release CMP and 2C-methyl-d-erythritol-2,4-cyclodiphosphate (MECP). The two least understood enzymes/steps are described together. The cyclodiphosphate, in two stages, undergoes a reduction and elimination to 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate and then on to IPP and DMAPP (32Rohdich F. Bacher A. Eisenreich W. Bioorg. Chem. 2004; 32: 292-308Crossref PubMed Scopus (67) Google Scholar). IspG transforms MECP to 2-methyl-2-(E)-butenyl diphosphate in a two-electron reduction. IspH then catalyzes production of IPP and some DMAPP. IspG and IspH both contain an iron-sulfur cluster, which explains their oxygen sensitivity. Under anaerobic conditions, catalysis is supported by a combination of NADPH, flavodoxin reductase, and flavodoxin. A flavodoxin has been implicated in the DOXP pathway and may be a physiological redox partner for one or both enzymes here (33Puan K.J. Wang H. Dairi T. Kuzuyama T. Morita C.T. FEBS Lett. 2005; 579: 3802-3806Crossref PubMed Scopus (75) Google Scholar). As of yet there are no structures for these enzymes, and further studies will be required to delineate specificity and mechanism. Isomerization of the IPP C=C bond to form DMAPP creates an electrophile exploited in reactions that provide diversity downstream in the biosynthetic routes that branch off from the non-mevalonate pathway. Two IPP isomerases, IDI-I and -II, maintain the supply of DMAPP. IDI-I is dependent on divalent cations, and structures of E. coli and human enzymes have been determined (34Durbecq V. Sainz G. Oudjama Y. Clantin B. Bompard-Gilles C. Tricot C. Caillet J. Stalon V. Droogmans L. Villeret V. EMBO J. 2001; 20: 1530-1537Crossref PubMed Scopus (83) Google Scholar, 35Wouters J. Oudjama Y. Barkley S.J. Tricot C. Stalon V. Droogmans L. Poulter C.D. J. Biol. Chem. 2003; 278: 11903-11908Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 36Zheng W. Sun F. Bartlam M. Li X. Li R. Rao Z. J. Mol. Biol. 2006; 366: 1447-1458Crossref PubMed Scopus (24) Google Scholar, 37Zhang C. Liu L. Xu H. Wei Z. Wang Y. Lin Y. Gong W. J. Mol. Biol. 2006; 366: 1437-1446Crossref PubMed Scopus (20) Google Scholar). IDI-II, which is restricted to the archaea and eubacteria, requires a metal ion, FMN, and (under aerobic conditions) NADPH. Structures of the Bacillus subtilis and T. thermophilus enzymes are published (38Steinbacher S. Kaiser J. Gerhardt S. Eisenreich W. Huber R. Bacher A. Rohdich F. J. Mol. Biol. 2003; 329: 973-982Crossref PubMed Scopus (51) Google Scholar, 39de Ruyck J. Rothman S.C. Poulter C.D. Wouters J. Biochem. Biophys. Res. Commun. 2005; 338: 1515-1518Crossref PubMed Scopus (28) Google Scholar). IDI-I is a compact α/β-protein. A flexible N-terminal segment becomes structured in the presence of a divalent cation (Mn2+ or Mg2+), helping to form the active site in a deep polar cavity. The ordering creates the allyl and diphosphate recognition parts of the active site. Three important residues (Cys-67, Tyr-10, and Glu-116 in EcIDI-I) participate in protonation and deprotonation of the isoprenoid. However, the contribution of each residue to the mechanism remains to be proven. The IDI-II subunit displays the triosephosphate isomerasetype α/β8 fold and oligomerizes to a cagelike octamer. Although dependent on several ligands, the contribution of each to catalysis remains unclear. Regulation of a metabolic pathway often depends on controlling levels of products within the pathway or of effector molecules. Covalent modification, exemplified by phosphorylation of HMG-CoA reductase can regulate an enzyme and influence activities downstream. There is also control by repression or activation of gene expression. In Gram-positive bacteria the MVA pathway genes are organized into operons and likely regulated by transcription. Compartmentation of enzymes can influence pathways, and we note that the MVA pathway occurs in the cytosol and mitochondria of plants whereas the DOXP pathway is in chloroplasts. The genes encoding the DOXP pathway enzymes are dispersed throughout genomes with no evidence of a global transcriptional regulator. However, ispD and ispF are transcriptionally coupled or fused, encoding a bifunctional enzyme. IspDF is unusual because, unlike most bifunctional enzymes, it catalyzes non-consecutive steps. The structure of the hexameric C. jejuni IspDF and associations of bifunctional and monofunctional enzymes (EcIspD, EcIspE, and EcIspF) have been investigated (23Gabrielsen M. Bond C.S. Hallyburton I. Hecht S. Bacher A. Eisenreich W. Rohdich F. Hunter W.N. J. Biol. Chem. 2004; 279: 52753-52761Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). A complex comprising three IspD and IspE dimers together with two IspF trimers creates an assembly localizing 18 catalytic centers. This could support efficient catalysis of three reactions at the core of isoprenoid precursor biosynthesis. There is, however, no evidence for enhanced catalytic rates on complex formation or any proof of substrate channeling (40Gabrielsen M. Rohdich F. Eisenreich W. Grawert T. Hecht S. Bacher A. Hunter W.N. Eur. J. Biochem. 2004; 271: 3028-3035Crossref PubMed Scopus (38) Google Scholar, 41Lherbet C. Pojer F. Richard S.B. Noel J.P. Poulter C.D. Biochemistry. 2006; 45: 3548-3553Crossref PubMed Scopus (19) Google Scholar). IspF binds isoprenoids in a conserved hydrophobic core created by trimer formation. Crystallographic studies have revealed the detail of IspF-isoprenoid interactions, and NMR combined with mass spectrometry shows that IspF binds phosphate, IPP or DMAPP, geranyl pyrophosphate, and farnesyl pyrophosphate (31Ni S. Robinson H. Marsing G.C. Bussiere D.E. Kennedy M.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1949-1957Crossref PubMed Scopus (24) Google Scholar, 42Kemp L.E. Alphey M.S. Bond C.S. Ferguson M.A. Hecht S. Bacher A. Eisenreich W. Rohdich F. Hunter W.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 45-52Crossref PubMed Scopus (40) Google Scholar). These ligands are synthesized two, three, or four enzymes downstream of IspF (43Kellogg B.A. Poulter C.D. Curr. Opin. Chem. Biol. 1997; 1: 570-578Crossref PubMed Scopus (162) Google Scholar) which raises the possibility of feedback regulation with IspF as a point of control. Enzymes of the DOXP pathway present attractive targets for development of broad spectrum antimicrobial drugs targeting serious diseases, including malaria, tuberculosis, and a range of sexually transmitted conditions. The DOXP pathway is absent from humans and occurs in serious human pathogens, and component enzymes have been validated genetically as drug targets (44Kobayashi K. Ehrlich S.D. Albertini A. Amati G. Andersen K.K. Arnaud M. Asai K. et al.Proc. Natl. Acad. U. S. A. 2003; 100: 4678-4683Crossref PubMed Scopus (1152) Google Scholar, 45Akerley B.J. Rubin E.J. Novick V.L. Amaya K. Judson N. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 966-971Crossref PubMed Scopus (327) Google Scholar, 46Sassetti C.M. Boyd D.H. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12712-12717Crossref PubMed Scopus (495) Google Scholar). 3Essential genes of E. coli were extracted by using a National Institute of Genetics (Japan) web site (www.shigen.nig.ac.jp/ecoli/pec/index.jsp) with information collated from a large number of related references. Enzymes such as these are particularly valued for drug development because they uniquely consume a substrate or generate a specific product, and their function cannot be compensated for by another enzyme (47Hasan S. Daugelat S. Rao P.S.S. Schreiber M. PLOS Comput. Biol. 2006; 6: 1-12Google Scholar). The DOXP enzymes are present in all intraerythrocytic stages of P. falciparum (48Cassera M.B. Gozzo F.C. D'Alexandri F.L. Merino E.F. del Portillo H.A. Peres V.J. Almeida I.C. Eberlin M.N. Wunderlich G. Wiesner J. Jomaa H. Kimura E.A. Katzin A.M. J. Biol. Chem. 2004; 279: 51749-51759Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and are nuclear encoded but transported to the apicoplast, a specialized non-photosynthetic organelle. The discovery of this relict chloroplast in this important parasite was exciting because the metabolic processes harbored within it, including isoprenoid biosynthesis, are of cyanobacterial origin and absent from mammals suggesting avenues for therapeutic intervention (49Ralph S.A. van Dooren G.G. Waller R.F. Crawford M.J. Fraunholz M.J. Foth B.J. Tonkin C.J. Roos D.S. McFadden G.I. Nat. Rev. Microbiol. 2004; 2: 203-216Crossref PubMed Scopus (512) Google Scholar). Comparisons indicate that key residues for substrate or cofactor binding in the DOXP enzymes are strictly conserved across species (50Kemp L.E. Bond C.S. Hunter W.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 607-610Crossref PubMed Scopus (33) Google Scholar) and that, with one exception, the E. coli enzyme structures represent templates to be exploited in structure-based approaches to drug development. The exception is IDI-I, which is present in E. coli and humans. It is IDI-II, by virtue of being very different from human IDI-I, that represents a therapeutic target. The DOXP enzymes also represent targets for herbicide development because inhibition would prevent chloroplast function for example. Also important is the potential for exploiting the pathway in designed biosynthesis. The DOXP pathway provides the route to the vast majority of plant terpenes (7Rodriguez-Concepcion M. Boronat A. Plant Physiol. 2002; 130: 1079-1089Crossref PubMed Scopus (655) Google Scholar, 51Eisenreich W. Rohdich F. Bacher A. Trends Plant Sci. 2001; 6: 78-84Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar), including the secondary metabolites that contribute to interactions with other organisms during pollination or seed dispersal or by providing protection from herbivores and pathogens. Such molecules are important for oils and flavors and are of medical importance. Consider taxol and artemisinin used to treat cancer and malaria, respectively, but which are only available from natural sources at low levels and which present significant challenges for industry scale synthesis. The exploitation of genetically modified organisms has exemplified ways to access such materials (52Martin V.J.J. Pitera D.J. Withers S.T. Newman J.D. Keasling J.D. Nat. Biotechnol. 2003; 21: 796-802Crossref PubMed Scopus (1404) Google Scholar), and in the future we can envisage manipulation of the pathways and of enzymes downstream to provide improved sources of isoprenoids.