Publications (22)122.48 Total impact
-
Article: Stimulation of Vgamma9/Vdelta2 T-lymphocyte proliferation by the isoprenoid precursor, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate.
[show abstract] [hide abstract]
ABSTRACT: (E)-1-Hydroxy-2-methyl-but-2-enyl 4-diphosphate, a recently discovered intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis, has been shown to act as a potent immunomodulator. In cultures of human peripheral blood mononuclear cells from eight non-related donors, the compound stimulated the proliferation of Vgamma9/Vdelta2 T lymphocytes with a median EC(50) of 70 pM when 10 U/ml of IL-2 was used as costimulant. Isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP) and some structural analogs of (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate also stimulated Vgamma9/Vdelta2 T-cell proliferation, albeit at much higher concentrations. The Vgamma9/Vdelta2 T-cell proliferation is highly dependent on the seeding density used in culture. All phosphoantigens tested elicited the proliferation of two T-lymphocyte populations with different apparent ratios between the expression level of Vdelta2 and Vgamma9 chains.Immunobiology 02/2007; 212(1):47-55. · 3.20 Impact Factor -
Article: The identification of isoprenoids that bind in the intersubunit cavity of Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase by complementary biophysical methods.
[show abstract] [hide abstract]
ABSTRACT: The discovery of a distinct metabolic pathway, the non-mevalonate or 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for isoprenoid precursor biosynthesis, in eubacteria and apicomplexan parasites has revealed a new set of potential drug targets. The emphasis of research on this pathway has been on delineating the intermediates and the biochemical and structural characterization of component enzymes. Two new monoclinic crystal forms of recombinant Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate (MECP) synthase cocrystallized with (i) CMP and (ii) CMP and MECP show well defined electron density at the subunit interface suggestive of an isoprenoid-like ligand. 31P NMR analysis of the recombinant protein sample indicates the presence of bound diphosphate species and electrospray mass spectrometry identifies a mixture of isopentenyl diphosphate (and/or dimethylallyl diphosphate), geranyl diphosphate and farnesyl diphosphate in an approximate ratio of 1:4:2. The most prevalent species, geranyl diphosphate, was successfully modelled into the electron density, revealing the important protein-ligand interactions that stabilize binding of the isoprenoid. The observation that MECP synthase binds three metabolites that are produced by enzymes two, three and four stages downstream in isoprenoid biosynthesis suggests that feedback regulation of the non-mevalonate pathway is possible.Acta Crystallographica Section D Biological Crystallography 02/2005; 61(Pt 1):45-52. · 12.62 Impact Factor -
Article: Hexameric assembly of the bifunctional methylerythritol 2,4-cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-dependent pathway of isoprenoid precursor biosynthesis.
[show abstract] [hide abstract]
ABSTRACT: The bifunctional methylerythritol 4-phosphate cytidylyltransferase methylerythritol 2,4-cyclodiphosphate synthase (IspDF) is unusual in that it catalyzes nonconsecutive reactions in the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway of isoprenoid precursor biosynthesis. The crystal structure of IspDF from the bacterial pathogen Campylobacter jejuni reveals an elongated hexamer with D3 symmetry compatible with the dimeric 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase and trimeric 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase monofunctional enzymes. Complex formation of IspDF with 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), the intervening enzyme activity in the pathway, has been observed in solution for the enzymes from C. jejuni and Agrobacterium tumefaciens. The monofunctional enzymes (2C-methyl-D-erythritol-4-phosphate cytidylyltransferase, IspE, and 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase) involved in the DOXP biosynthetic pathway of Escherichia coli also show physical associations. We propose that complex formation of the three enzymes at the core of the DOXP pathway can produce an assembly localizing 18 catalytic centers for the early stages of isoprenoid biosynthesis.Journal of Biological Chemistry 01/2005; 279(50):52753-61. · 4.77 Impact Factor -
Article: IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis.
[show abstract] [hide abstract]
ABSTRACT: The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.Journal of the American Chemical Society 11/2004; 126(40):12847-55. · 9.91 Impact Factor -
Article: Biochemical characterization of Bacillus subtilis type II isopentenyl diphosphate isomerase, and phylogenetic distribution of isoprenoid biosynthesis pathways.
[show abstract] [hide abstract]
ABSTRACT: An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184,997-186,043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 micromol.mg(-1).min(-1), respectively, as diagnosed by 1H spectroscopy. FMN and divalent cations are required for catalytic activity; the highest rates were found with Ca2+. NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of (S)-alpha-hydroxyacid oxidizing enzymes including flavocytochrome b2 and L-lactate dehydrogenase and was shown to catalyse the formation of [2,3-13C2]lactate from [2,3-13C2]pyruvate, albeit at a low rate of 1 nmol.mg(-1).min(-1). Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy.European Journal of Biochemistry 08/2004; 271(13):2658-69. · 3.58 Impact Factor -
Article: Biosynthesis of isoprenoids: a bifunctional IspDF enzyme from Campylobacter jejuni.
[show abstract] [hide abstract]
ABSTRACT: In the nonmevalonate pathway of isoprenoid biosynthesis, the conversion of 2C-methyl-d-erythritol 4-phosphate into its cyclic diphosphate proceeds via nucleotidyl intermediates and is catalyzed by the products of the ispD, ispE and ispF genes. An open reading frame of Campylobacter jejuni with similarity to the ispD and ispF genes of Escherichia coli was cloned into an expression vector directing the formation of a 42 kDa protein in a recombinant E. coli strain. The purified protein was shown to catalyze the transformation of 2C-methyl-D-erythritol 4-phosphate into 4-diphosphocytidyl-2C-methyl-D-erythritol and the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate at catalytic rates of 19 micro mol x mg(-1) x min(-1) and 7 micro mol x mg(-1) x min(-1), respectively. Both enzyme-catalyzed reactions require divalent metal ions. The C. jejuni enzyme does not catalyze the formation of 2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol, a side reaction catalyzed in vitro by the IspF proteins of E. coli and Plasmodium falciparum. Comparative genomic analysis show that all sequenced alpha- and epsilon-proteobacteria have fused ispDF genes. These bifunctional proteins are potential drug targets in several human pathogens (e.g. Helicobacter pylori, C. jejuni and Treponema pallidum).European Journal of Biochemistry 08/2004; 271(14):3028-35. · 3.58 Impact Factor -
Article: Biosynthesis of isoprenoids
[show abstract] [hide abstract]
ABSTRACT: In the nonmevalonate pathway of isoprenoid biosynthesis, the conversion of 2C-methyl-d-erythritol 4-phosphate into its cyclic diphosphate proceeds via nucleotidyl intermediates and is catalyzed by the products of the ispD, ispE and ispF genes. An open reading frame of Campylobacter jejuni with similarity to the ispD and ispF genes of Escherichia coli was cloned into an expression vector directing the formation of a 42 kDa protein in a recombinant E. coli strain. The purified protein was shown to catalyze the transformation of 2C-methyl-d-erythritol 4-phosphate into 4-diphosphocytidyl-2C-methyl-d-erythritol and the conversion of 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate into 2C-methyl-d-erythritol 2,4-cyclodiphosphate at catalytic rates of 19 µmol·mg−1·min−1 and 7 µmol·mg−1·min−1, respectively. Both enzyme-catalyzed reactions require divalent metal ions. The C. jejuni enzyme does not catalyze the formation of 2C-methyl-d-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-d-erythritol, a side reaction catalyzed in vitro by the IspF proteins of E. coli and Plasmodium falciparum. Comparative genomic analysis show that all sequenced α- and ε-proteobacteria have fused ispDF genes. These bifunctional proteins are potential drug targets in several human pathogens (e.g. Helicobacter pylori, C. jejuni and Treponema pallidum).European Journal of Biochemistry. 06/2004; 271(14):3028 - 3035. -
Article: Biosynthetic experiments with tall plants under field conditions. 18O2 incorporation into humulone from Humulus lupulus.
[show abstract] [hide abstract]
ABSTRACT: Five segments of a large hop plant (Humulus lupulus var. Hallertauer Magnum) carrying several cones were enclosed in sealed glass vessels that were gassed with (18)O(2). After 14 days, the segments were harvested and humulone and cohumulone were analysed by NMR spectroscopy and mass spectroscopy. The oxygen atoms in position 6 of humulone and cohumulone showed 9% (18)O enrichment, respectively. It follows that the C-6 hydroxy groups were introduced by oxygenase catalysis.Phytochemistry 05/2004; 65(8):1057-60. · 3.35 Impact Factor -
Article: A possible primordial peptide cycle.
[show abstract] [hide abstract]
ABSTRACT: alpha-Amino acids can undergo peptide formation by activation with carbon monoxide (CO) under hot aqueous conditions in the presence of freshly coprecipitated colloidal (Fe,Ni)S. We now show that CO-driven peptide formation proceeds concomitantly with CO-driven, N-terminal peptide degradation by racemizing N-terminal hydantoin and urea derivatives to alpha-amino acids. This establishes a peptide cycle with closely related anabolic and catabolic segments. The hydantoin derivative is a purin-related heterocycle. The (Fe,Ni)S-dependent urea hydrolysis could have been the evolutionary precursor of the nickelenzyme urease. The results support the theory of a chemoautotrophic origin of life with a CO-driven, (Fe,Ni)S-dependent primordial metabolism.Science 09/2003; 301(5635):938-40. · 31.20 Impact Factor -
Article: Biosynthesis of isoprenoids: crystal structure of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase.
[show abstract] [hide abstract]
ABSTRACT: 4-Diphosphocytidyl-2C-methyl-d-erythritol kinase, an essential enzyme in the nonmevalonate pathway of isopentenyl diphosphate and dimethylallyl diphosphate biosynthesis, catalyzes the single ATP-dependent phosphorylation stage affording 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate. The 2-A resolution crystal structure of the Escherichia coli enzyme in a ternary complex with substrate and a nonhydrolyzable ATP analogue reveals the molecular determinants of specificity and catalysis. The enzyme subunit displays the alpha/beta fold characteristic of the galactose kinase/homoserine kinase/mevalonate kinase/phosphomevalonate kinase superfamily, arranged into cofactor and substrate-binding domains with the catalytic center positioned in a deep cleft between domains. Comparisons with related members of this superfamily indicate that the core regions of each domain are conserved, whereas there are significant differences in the substrate-binding pockets. The nonmevalonate pathway is essential in many microbial pathogens and distinct from the mevalonate pathway used by mammals. The high degree of sequence conservation of the enzyme across bacterial species suggests similarities in structure, specificity, and mechanism. Our model therefore provides an accurate template to facilitate the structure-based design of broad-spectrum antimicrobial agents.Proceedings of the National Academy of Sciences 09/2003; 100(16):9173-8. · 9.68 Impact Factor -
Article: The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein.
[show abstract] [hide abstract]
ABSTRACT: Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-d-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol x min(-1) x mg(-1). Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 micromol x min(-1) x mg(-1). IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol x min(-1) x mg(-1). The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.Proceedings of the National Academy of Sciences 03/2003; 100(4):1586-91. · 9.68 Impact Factor -
Article: Deoxyxylulose phosphate pathway of isoprenoid biosynthesis. Discovery and function of ispDEFGH genes and their cognate enzymes
[show abstract] [hide abstract]
ABSTRACT: Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) serve as the universal precursors for the biosynthesis of terpenes. Besides the well-known mevalonate pathway, a second biosynthetic pathway conducive to IPP and DMAPP via 1-deoxy-D-xylu-lose-5-phosphate and 2C-methyl-D-erythritol-4-phosphate has been discovered recently in plants and certain eubacteria. 2C-Methyl-D-erythritol-4-phosphate, the first committed inter-mediate of the deoxyxylulose phosphate pathway, is converted into 2C-methyl-D-erythritol 2,4-cyclodiphosphate by the catalytic action of three enzymes specified by the ispDEF genes. The cyclic diphosphate is reductively opened by the IspG protein affording 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate. This compound can be converted into IPP as well as DMAPP by the catalytic action of IspH protein. The enzymes of this pathway are potential targets for novel antibacterial, antimalarial, and herbicide agents.Pure Appl. Chem. 01/2003; 75:2-3. -
Article: Biosynthesis of terpenes: studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase.
[show abstract] [hide abstract]
ABSTRACT: Earlier in vivo studies showed the involvement of IspH protein in the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). We have demonstrated now that cell extract of an Escherichia coli strain engineered for hyperexpression of the ispH (lytB) gene catalyzes the in vitro conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into IPP and DMAPP. The reaction requires NADH, FAD, divalent cations (preferably Co2+), and probably one or more as-yet-unidentified proteins. The low intrinsic catalytic activities of wild-type E. coli cell extract and isolated chromoplasts of red pepper (Capsicum annuum) are enhanced by the addition of purified recombinant IspH protein.Proceedings of the National Academy of Sciences 10/2002; 99(19):12108-13. · 9.68 Impact Factor -
Article: Biosynthesis of terpenes. Preparation of (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate, an intermediate of the deoxyxylulose phosphate pathway.
[show abstract] [hide abstract]
ABSTRACT: (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (E-6) was synthesized in six reaction steps from hydroxyacetone (9) and (ethoxycarbonylmethenyl)-triphenylphosphorane (11) with an overall yield of 38%. The compound was shown to be identical with the product of IspG protein, which serves as an intermediate in the nonmevalonate terpene biosynthetic pathway.The Journal of Organic Chemistry 07/2002; 67(13):4590-4. · 4.45 Impact Factor -
Article: Studies on the nonmevalonate terpene biosynthetic pathway: metabolic role of IspH (LytB) protein.
[show abstract] [hide abstract]
ABSTRACT: Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes. Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy-D-xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria. Earlier in vivo experiments with recombinant Escherichia coli strains showed that exogenous 1-deoxy-D-xylulose can be converted into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of enzymes specified by the xylB and ispCDEFG genes. This article describes the transformation of exogenous [U-(13)C(5)]1-deoxy-D-xylulose into a 5:1 mixture of [U-(13)C(5)]isopentenyl diphosphate and [U-(13)C(5)]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.Proceedings of the National Academy of Sciences 03/2002; 99(3):1158-63. · 9.68 Impact Factor -
Article: Structure of 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase involved in mevalonate-independent biosynthesis of isoprenoids.
[show abstract] [hide abstract]
ABSTRACT: Isoprenoids are biosynthesized from isopentenyl diphosphate and the isomeric dimethylallyl diphosphate via the mevalonate pathway or a mevalonate-independent pathway that was identified during the last decade. The non-mevalonate pathway is present in many bacteria, some algae and in certain protozoa such as the malaria parasite Plasmodium falciparum and in the plastids of higher plants, but not in mammals and archaea. Therefore, these enzymes have been recognised as promising drug targets. We report the crystal structure of Escherichia coli 2C- methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), which converts 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate into 2C-methyl-d-erythritol 2,4-cyclodiphosphate and CMP in a Mg-dependent reaction. The protein forms homotrimers that tightly bind one zinc ion per subunit at the active site, which helps to position the substrate for direct attack of the 2-phosphate group on the beta-phosphate.Journal of Molecular Biology 03/2002; 316(1):79-88. · 4.00 Impact Factor -
Article: Studies on the nonmevalonate pathway of terpene biosynthesis
[show abstract] [hide abstract]
ABSTRACT: 2C-Methyl-d-erythritol 2,4-cyclodiphosphate was recently shown to be formed from 2C-methyl-d-erythritol 4-phosphate by the consecutive action of IspD, IspE, and IspF proteins in the nonmevalonate pathway of terpenoid biosynthesis. To complement previous work with radiolabelled precursors, we have now demonstrated that [U-13C5]2C-methyl-d-erythritol 4-phosphate affords [U-13C5]2C-methyl-d-erythritol 2,4-cyclodiphosphate in isolated chromoplasts of Capsicum annuum and Narcissus pseudonarcissus. Moreover, chromoplasts are shown to efficiently convert 2C-methyl-d-erythritol 4-phosphate as well as 2C-methyl-d-erythritol 2,4-cyclodiphosphate into the carotene precursor phytoene. The bulk of the kinetic data collected in competition experiments with radiolabeled substrates is consistent with the notion that the cyclodiphosphate is an obligatory intermediate in the nonmevalonate pathway to terpenes. Studies with [2,2′-13C2]2C-methyl-d-erythritol 2,4-cyclodiphosphate afforded phytoene characterized by pairs of jointly transferred 13C atoms in the positions 17/1, 18/5, 19/9, and 20/13 and, at a lower abundance, in positions 16/1, 4/5, 8/9, and 12/13. A detailed scheme is presented for correlating the observed partial scrambling of label with the known lack of fidelity of the isopentenyl diphosphate/dimethylethyl diphosphate isomerase.European Journal of Biochemistry. 12/2001; 268(23):6302 - 6310. -
Article: Studies on the Non‐Mevalonate Pathway − Preparation and Properties of Isotope‐Labeled 2C‐Methyl‐D‐erythritol 2,4‐Cyclodiphosphate
[show abstract] [hide abstract]
ABSTRACT: Recent studies have shown that 2C-methyl-D-erythritol 2,4-cyclodiphosphate can be formed from 2C-methyl-D-erythritol 4-phosphate by the consecutive action of IspD, IspE and IspF proteins in the non-mevalonate pathway of isoprenoid biosynthesis. We describe here rapid one-pot strategies for the enzyme-assisted preparation of this compound from isotope-labeled pyruvate and glucose that are optimized for the introduction of 13C or 14C and afford a wide variety of isotopomers in mmol quantity. The one-pot reactions involving up to 10 forward reaction steps and up to 15 enzyme catalysts have an overall yield of 50 to 80% of purified product.Annalen der Chemie und Pharmacie 08/2001; 2001(17):3221 - 3226. · 3.10 Impact Factor -
Article: Biosynthesis of terpenoids
[show abstract] [hide abstract]
ABSTRACT: The putative catalytic domain of an open reading frame from Plasmodium falciparum with similarity to the ispF gene of Escherichia coli specifying 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase was expressed in a recombinant E. coli strain. The recombinant protein was purified to homogeneity and was found to catalyze the formation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate from 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate at a rate of 4.3 µmol·mg−1·min−1. At lower rates, the recombinant protein catalyzes the formation of 2-phospho-2C-methyl-d-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate and the formation of 2C-methyl-d-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-d-erythritol. Divalent metal ions such as magnesium or manganese are required for catalytic activity. The enzyme has a pH optimum at pH 7.0. Recombinant expression of the full-length open reading frame afforded insoluble protein that could not be folded in vitro. The enzyme is a potential target for antimalarial drugs directed at the nonmevalonate pathway of isoprenoid biosynthesis.European Journal of Biochemistry. 05/2001; 268(11):3190 - 3197. -
Article: Biosynthesis of Isoprenoids. A Rapid Method for the Preparation of Isotope-Labeled 4-Diphosphocytidyl-2C-methyl-d-erythritol
[show abstract] [hide abstract]
ABSTRACT: 4-Diphosphocytidyl-2C-methyl-d-erythritol serves as an intermediate in the nonmevalonate pathway of isoprenoid biosynthesis. The compound has been prepared in millimole quantity by a sequence of one-pot reactions using 13C-labeled pyruvate and dihydroxyacetone phosphate or 13C-labeled glucose as starting materials and recombinant enzymes of the nonmevalonate isoprenoid pathway as catalysts. The method has been used for the preparation of various 4-diphosphocytidyl-2C-methyl-d-erythritol isotopomers in high yield.09/2000;
Top co-authors
Top Journals
Institutions
-
2002–2007
-
Technische Universität München
- Biochemistry
München, Bavaria, Germany -
Max-Planck-Institut für Biochemie
- Abteilung für Strukturforschung
München, Bavaria, Germany
-
-
2003–2005
-
University of Dundee
- College of Life Sciences
Dundee, SCT, United Kingdom
-
