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Crystal Structure of 4-Diphosphocytidyl-2C-Methyl-D-Erythritol Kinase

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Crystal Structure of 4-Diphosphocytidyl-2C-Methyl-D-Erythritol Kinase Biosynthesis of isoprenoids: Crystal structure of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase Linda Miallau*†, Magnus S. Alphey*, Lauris E. Kemp*, Gordon A. Leonard†, Sean M. McSweeney†, Stefan Hecht‡, Adelbert Bacher‡, Wolfgang Eisenreich‡, Felix Rohdich‡, and William N. Hunter*§ *Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom; †Macromolecular Crystallography Group, European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble Cedex 9, France; and ‡Institut fu¨r Organische Chemie und Biochemie, Technische Universita¨t Mu¨ nchen, Lichtenbergstrasse 4, D-85747 Garching, Germany Communicated by Duilio Arigoni, Swiss Federal Institute of Technology, Zu¨rich, Switzerland, June 5, 2003 (received for review May 1, 2003) 4-Diphosphocytidyl-2C-methyl-D-erythritol kinase, an essential en- 8). The first stage in the nonmevalonate route is the condensa- zyme in the nonmevalonate pathway of isopentenyl diphosphate tion of pyruvate and D-glyceraldehyde 3-phosphate to produce and dimethylallyl diphosphate biosynthesis, catalyzes the single DOXP (9, 10). The second and third stages convert DOXP to ATP-dependent phosphorylation stage affording 4-diphosphocyti- 2C-methyl-D-erythritol-4-phosphate (11, 12) and transfer the dyl-2C-methyl-D-erythritol-2-phosphate. The 2-Å resolution crystal erythritol derivative onto a nucleotide, resulting in CDP-ME structure of the Escherichia coli enzyme in a ternary complex with (13–15). Stage four, the only ATP-dependent step in the path- substrate and a nonhydrolyzable ATP analogue reveals the molec- way, is catalyzed by CDP-ME kinase (16, 17), the subject of the ular determinants of specificity and catalysis. The enzyme subunit present study. Here, CDP-ME kinase catalyzes the transfer of displays the ␣͞␤ fold characteristic of the galactose kinase͞ the ␥-phosphoryl moiety of ATP to CDP-ME, forming 4-diphos- ͞ ͞ homoserine kinase mevalonate kinase phosphomevalonate ki- phocytidyl-2C-methyl-D-erythritol-2-phosphate (CDP-ME2P) nase superfamily, arranged into cofactor and substrate-binding and ADP (Fig. 1). Next, CDP-ME2P is converted to 2C-methyl- domains with the catalytic center positioned in a deep cleft D-erythritol-2,4-cyclodiphosphate and CMP (18, 19). The cyclo- between domains. Comparisons with related members of this diphosphate then, in two enzyme-catalyzed stages, undergoes superfamily indicate that the core regions of each domain are reduction and elimination to 1-hydroxy-2-methyl-2-(E)-butenyl- conserved, whereas there are significant differences in the sub- 4-diphosphate and onto IPP and DMAPP (20–23). strate-binding pockets. The nonmevalonate pathway is essential in The species that use the DOXP pathway include the causal many microbial pathogens and distinct from the mevalonate path- agents for diverse and serious human diseases including leprosy, way used by mammals. The high degree of sequence conservation malaria, bacterial meningitis, various gastrointestinal and sexu- of the enzyme across bacterial species suggests similarities in ally transmitted infections, tuberculosis, and certain types of structure, specificity, and mechanism. Our model therefore pro- pneumonia (5). The enzymes of the DOXP pathway have no vides an accurate template to facilitate the structure-based design orthologs in humans and have attracted interest as potential of broad-spectrum antimicrobial agents. targets for structure-based antimicrobial drug development. DOXP reductoisomerase, the enzyme that catalyzes the second galactose kinase͞homoserine kinase͞mevalonate kinase͞ reaction in the pathway, is inhibited by the antibiotic fosmido- phosphomevalonate kinase ͉ enzyme mechanism ͉ mycin (24, 25), a compound that has performed well in recent nonmevalonate ͉ phosphorylation clinical trials against malaria (26). Such chemical validation of the reductoisomerase is complemented by genetic studies, which he ATP-dependent 4-diphosphocytidyl-2C-methyl-D- have proven that the enzymes responsible for stages three to five Terythritol (CDP-ME) kinase (EC 2.7.1.148) participates in of the pathway are essential in eubacteria (27–31). The crystal- the biosynthesis of isopentenyl diphosphate (IPP) and dimethy- lographic studies necessary to support structure-based drug lallyl diphosphate (DMAPP). These isomers are the universal discovery have produced models for DOXP reductoisomerase five-carbon precursors of isoprenoids, a diverse and important (32–34), 2C-methyl-D-erythritol-4-phosphate cytidylyltrans- family of natural products that includes sterols, dolichols, trit- ferase (15, 35), and 2C-methyl-D-erythritol-2,4-cyclodiphos- erpenes, and ubiquinones, and components of macromolecules phate synthase (36–39). We now turn our attention to CDP-ME such as the prenyl groups of prenylated proteins and isopente- kinase. nylated tRNAs (1–3). Isoprenoids contribute to many biological On the basis of sequence comparisons, CDP-ME kinase is functions, including electron transport in respiration and pho- recognized as a member of the galactose kinase͞homoserine BIOCHEMISTRY tosynthesis, hormone-based signaling, apoptosis, meiosis, pro- kinase (HSK)͞mevalonate kinase͞phosphomevalonate kinase tein cleavage, and degradation (4). In addition, they provide (GHMP) kinase superfamily (40), so-called after the four found- important structural components of cell membranes (1). ing members, namely galacto, homoserine, mevalonate, and Two biosynthetic routes to IPP and DMAPP have evolved. In phosphomevalonate kinases (41). The GHMP family now also eukaryotes, archaebacteria, and a few eubacteria, the precursor includes mevalonate 5-diphosphate decarboxylase (42) and ar- biosynthesis is through the mevalonate pathway (3–7). This chaeal shikimate kinase (43). Crystal structures are available for begins with the conversion of three molecules of acetyl-CoA to several GHMP kinases (40, 42, 44–47) although only HSK (38) 3-hydroxy-3-methylglutaryl-CoA followed by reduction, phos- has provided experimental details for a ternary complex. In phorylation, and decarboxylation to generate IPP, some of which is isomerized to DMAPP. The last three steps in this pathway are ATP-dependent and catalyzed by the structurally related meva- Abbreviations: AMP-PNP, adenosine 5Ј-(␤,␥-imino)triphosphate; CDP-ME, 4-diphospho- lonate kinase (MVK), phosphomevalonate kinase, and meval- cytidyl-2C-methyl-D-erythritol; DMAPP, dimethylallyl diphosphate; DOXP, 1-deoxy-D- xylulose-5-phosphate; GHMP, galactose kinase͞homoserine kinase͞mevalonate kinase͞ onate 5-diphosphate decarboxylase. phosphomevalonate kinase; HSK, homoserine kinase; IPP, isopentenyl diphosphate; MVK, In chloroplasts, algae, cyanobacteria, most eubacteria, and the mevalonate kinase; NCS, noncrystallographic symmetry. apicomplexa, IPP and DMAPP synthesis is accomplished by Data deposition: The atomic coordinates and structure factors have been deposited in the seven enzymes in a pathway named after one of the intermedi- Protein Data Bank, www.rcsb.org (PDB ID code 10J4). ates, the 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway (5– §To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.1533425100 PNAS ͉ August 5, 2003 ͉ vol. 100 ͉ no. 16 ͉ 9173–9178 Downloaded by guest on September 30, 2021 Table 1. Data collection and refinement statistics Resolution, Å͞completeness 2.0͞94.3 (92.3) No. of measurements͞unique reflections 364,197͞39,761 I͞␴(I)͞Rmerge,% 12.9 (5.6)͞4.8 (14.7) Protein residues͞water molecules 566͞383 AMP-PNP͞CDP-ME͞ClϪ 2͞2͞1 Rwork,%͞no. of reflections 16.6͞37,773 Rfree,%͞no. of reflections 20.2͞1,988 Wilson B, Å2 17.8 Average isotropic thermal parameters, Å2 Main chain͞side chain 15.3͞18.4 Solvents͞AMP-PNP͞CDP-ME 28.9͞44.0͞29.1 rms deviation for bond lengths, Å͞bond angles, ° 0.010͞1.33 Ramachandran analysis, % Favored regions͞additionally allowed regions 92.9͞7.1 Numbers in parentheses refer to a high-resolution bin of approximate width 0.1 Å. SDS͞PAGE and matrix-assisted laser desorption time-of-flight MS; the latter technique also confirmed full incorporation of L-selenomethionine. The enzyme was incubated with 2 mM AMP-PNP (Sigma) and 3 mM CDP-ME (48) on ice for2htoprovide a stock solution for crystallization. Monoclinic plate-like crystals grew in hanging drops consisting of 1 ␮l of stock, 1 ␮l of reservoir solution (20% polyethylene glycol 8000, 0.2 M magnesium acetate, 0.1 M sodium cacodylate, pH 6.5), and 0.2 ␮l of 0.25 M sulfo-betaine. Fig. 1. The reaction catalyzed by CDP-ME kinase. Data Collection, Structure Solution, and Refinement. Crystals were maintained at 100 K in the presence of 20% glycerol as a cryoprotectant, and measurements were made on beamline ID29 other cases modeling has been used to support mechanistic at the European Synchrotron Radiation Facility with an ADSC- studies (45, 46). Q210 charge-coupled device detector. A single crystal of L- To elucidate details of the enzyme structure, specificity, and selenomethionine derivilized protein was used to measure 180° mechanism we have determined the high-resolution crystal of data at a wavelength (␭ 0.9791 Å), determined from a Se structure of recombinant Escherichia coli CDP-ME kinase in K-edge x-ray absorption near-edge scan, to maximize the dis- complex with substrate and the hydrolysis-resistant ATP ana- persive fЉ signal. A single-wavelength anomalous dispersion Ј ␤ ␥ logue adenosine 5 -( , -imino)triphosphate (AMP-PNP). Se- approach provided initial phases. quence and structural
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