Structure of 2C-methyl- d -erythritol 2,4- cyclodiphosphate synthase: An essential enzyme for isoprenoid biosynthesis and target for antimicrobial drug development

Abstract

The crystal structure of the zinc enzyme Escherichia coli 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase in complex with cytidine 5 -diphosphate and Mn 2؉ has been determined to 1.8-Å resolution. This enzyme is essential in E. coli and participates in the nonmevalonate pathway of isoprenoid biosynthesis, a critical pathway present in some bacterial and apicomplexans but distinct from that used by mammals. Our analysis reveals a homotrimer, built around a ␤ prism, carrying three active sites, each of which is formed in a cleft between pairs of subunits. Residues from two subunits recognize and bind the nucleotide in an active site that contains a Zn 2؉ with tetrahedral coordination. A Mn 2؉ , with octahedral geometry, is positioned between the ␣ and ␤ phosphates acting in concert with the Zn 2؉ to align and polarize the substrate for catalysis. A high degree of sequence conservation for the enzymes from E. coli, Plasmodium falciparum, and Mycobacterium tuberculosis suggests similarities in secondary structure, subunit fold, quaternary structure, and active sites. Our model will therefore serve as a template to facilitate the structurebased design of potential antimicrobial agents targeting two of the most serious human diseases, tuberculosis and malaria.zinc enzyme ͉ Escherichia coli ͉ Plasmodium falciparum ͉ Mycobacterium tuberculosis T he enzyme 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MECP) synthase participates in the biosynthesis of the isomers isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate. These compounds are the universal five-carbon precursors of isoprenoids, a diverse and large family of natural products including sterols, dolichols, triterpenes, ubiquinones, and plastoquinone, and also components of macromolecules such as the prenyl groups of prenylated proteins and isopentenylated tRNAs (1-3). Isoprenoids contribute to many functions, including electron transport in respiration and photosynthesis, hormone-based signaling, the regulation of transcription, and posttranslational processes that control lipid biosynthesis, meiosis, apoptosis, protein cleavage, and degradation. In addition, certain isoprenoids constitute an important structural component of cell membranes.Isoprenoid biosynthesis depends on the production of IPP, which occurs in mammals, higher plants, fungi, and certain bacteria through the mevalonate (MVA) pathway (4-7). This pathway begins with the conversion of acetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA, followed by reduction, phosphorylation, and decarboxylation to generate IPP, some of which is then isomerized to dimethylallyl pyrophosphate. The MVA pathway was considered ubiquitous until recently, when a nonmevalonate-dependent route, the 1-deoxy-D-xylulose 5-phosphate (DOXP) or 2C-methyl-D-erythritol 4-phosphate (MEP) pathway, was discovered in chloroplasts, algae, cyanobacteria, eubacteria, and apicomplexa (2, 6, 7).Five distinct steps of the DOXP͞MEP pathway have been elucidated (6-10). The pathway starts with the condensation of pyruvate and glyceralde...

Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate β352 as a Magnesium Ligand

“…Proposed Magnesium Binding Site-We speculate that the magnesium binding site of FTase probably resembles the metal binding sites found in GDP-mannose mannosyl hydrolase and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, where a divalent metal ion coordinates two nonbridging oxygens of the diphosphate ligand and one carboxylate side chain of the enzyme (49,50). In 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, the bound manganese ion is only 5.7 Å away from the active site zinc ion.…”

Biosynthesis of isoprenoids in plants: Structure of the 2C‐methyl‐d‐erithrytol 2,4‐cyclodiphosphate synthase from Arabidopsis thaliana. Comparison with the bacterial enzymes

“…Electron density had also been found inside the cavities of other reported MCS structures (Kemp et al 2002;Lehmann et al 2002;Richard et al 2002;Ni et al 2004). For the bacterial structures from E. coli (Richard et al 2002;Kemp et al 2005), S. oneidensis (Ni et al 2004), and C. jejuni (IspDF, a bifunctional enzyme); (Gabrielsen et al 2004), the electron density inside the cavity was interpreted as GPP or as farnesyl diphosphate (FPP) with the diphosphate moieties positioned along the molecular threefold axis and the a-phosphate group interacting with the arginines at the cavity entrance (Fig.…”

“…The b-sheets of the three subunits face one another, defining an internal b-barrel like structure that is surrounded by the a-helices on the external surface of the trimer. In the trimer, extensive inter-subunit interactions define a network of complementary hydrophobic contacts, electrostatic interactions, and specific hydrogen bonds, similarly to what was described for the bacterial MCS enzymes (Kemp et al 2002;Lehmann et al 2002;Richard et al 2002;Steinbacher et al 2002;Ni et al 2004). Multiple alignment of plant and bacterial MCS proteins.…”

“…The three-dimensional structures of five bacterial MCS enzymes have been reported: from E. coli (Kemp et al 2002;Richard et al 2002;Steinbacher et al 2002), Haemophilus influenzae (Lehmann et al 2002), the thermophile Thermus thermophilus (Kishida et al 2003), Shewanella oneidensis (Ni et al 2004), and the MCS domain of a bifunctional IspDF enzyme from Campylobacter jejuni (Gabrielsen et al 2004). In this work, we present the X-ray crystal structure of the MCS enzyme from A. thaliana, the first determined from a plant.…”

Biosynthesis of isoprenoids

“…13 C NMR spectra were recorded at intervals. Residues involved in substrate binding [31][32][33] are labeled by triangles; conserved residues are shown in grey, identical residues in black. Kinetic studies on recombinant IspDF protein by NMR spectroscopy 13 C NMR signal intensities were used to perform numerical simulations using the program DYNAFIT [30].…”