Nonmevalonate Terpene Biosynthesis Enzymes as Antiinfective Drug Targets:  Substrate Synthesis and High-Throughput Screening Methods

Illarionova, Victoria; Kaiser, J.; Ostrozhenkova, Elena; Bacher, Adelbert; Fischer, Markus; Eisenreich, Wolfgang; Rohdich, Felix

doi:10.1021/jo061466o

Cited by 52 publications

(72 citation statements)

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“…However, it appears safe to say that the present experiments extend the degree of stringency to the limits of experimental feasibility as ultimately defined by the long-term chemical stability of the proteins, substrates and coenzymes involved. C 3 ]2C-methyl-d-erythrose 4-phosphate were synthesized as described previously [37,43,44].…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of isoprenoids – studies on the mechanism of 2C‐methyl‐d‐erythritol‐4‐phosphate synthase

Lauw¹,

Illarionova²,

Bacher³

et al. 2008

The FEBS Journal

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2C‐Methyl‐d‐erythritol‐4‐phosphate synthase, encoded by the ispC gene (also designated dxr), catalyzes the first committed step in the nonmevalonate isoprenoid biosynthetic pathway. The reaction involves the isomerization of 1‐deoxy‐d‐xylulose 5‐phosphate, giving a branched‐chain aldose derivative that is subsequently reduced to 2C‐methyl‐d‐erythritol 4‐phosphate. The isomerization step has been proposed to proceed as an intramolecular rearrangement or a retroaldol–aldol sequence. We report the preparation of 13C‐labeled substrate isotopologs that were designed to optimize the detection of an exchange of putative cleavage products that might occur in the hypothetical retroaldol–aldol reaction sequence. In reaction mixtures containing large amounts of 2C‐methyl‐d‐erythritol‐4‐phosphate synthase from Escherichia coli, Mycobacterium tuberculosis or Arabidopsis thaliana, and a mixture of [1‐13C1]‐2C‐methyl‐d‐erythritol 4‐phosphate and [3‐13C1]2C‐methyl‐d‐erythritol 4‐phosphate, the reversible reaction could be followed over thousands of reaction cycles. No fragment exchange could be detected by NMR spectroscopy, and the frequency of exchange, if any, is less than 5 p.p.m. per catalytic cycle. Hydroxyacetone, the putative second fragment expected from the retroaldol cleavage, was not incorporated into the enzyme product. In contrast to other reports, IspC did not catalyze the isomerisation of 1‐deoxy‐d‐xylulose 5‐phosphate to give 1‐deoxy‐l‐ribulose 5‐phosphate under any conditions tested. However, we could show that the isomerization reaction proceeds at room temperature without a requirement for enzyme catalysis. Although a retroaldol–aldol mechanism cannot be ruled out conclusively, the data show that a retroldol–aldol reaction sequence would have to proceed with very stringent fragment containment that would apply to the enzymes from three genetically distant organisms.

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of isoprenoids – studies on the mechanism of 2C‐methyl‐d‐erythritol‐4‐phosphate synthase

Lauw¹,

Illarionova²,

Bacher³

et al. 2008

The FEBS Journal

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“…The settings of the instrument were: collision energy, -54 V; declustering potential, -40 V; cell entrance potential, -30 V; cell exit potential, -0 V; and entrance potential, -8 V. Both Q1 and Q2 quadrupoles were maintained at high mass resolution of approximately 0.5 D. Analyst 1.5 software (Applied Biosystems) was used for data acquisition and processing. The MEcDP content in the plant extracts were quantified using external standard curves and normalized to additionally added (3,4,(5)(6)(7)(8)(9)(10)(11)(12)(13) C)-MEcDP internal standard (ISTD; Illarionova et al, 2006). Normalization to added labeled standards was accomplished by analyzing each plant sample twice, once without any added ISTD and the second time with the addition of MEcDP ISTD dissolved in 10 mL water.…”

Section: Quantification and Isotopic 13 C Compositions Of Mecdp Dmadmentioning

confidence: 99%

Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene

et al. 2014

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The plastidic 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope 13 C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus 3 canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO 2 ]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-Dxylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-D-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. b-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties.

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“…The crude product was used in the next step without further purification and was not fully characterized. 1 3468w, 3310w, 2927w, 1624s, 1598m, 1543m, 1497s, 1417m, 1381m, 1278s, 1118w, 1090m [15]. IspF E. coli was prepared according to the procedure reported in [6].…”

Section: Experimental Partmentioning

confidence: 99%

“…The dependence of the intensity of the 13 C-signals of the substrate and the product (measured by integration) on the concentration of a given inhibitor was used to determine a % inhibition. Substrate/product ratios were obtained from the signals of C(1) of [1,3,4-13 C 3 ]-5 and C-1 of [1,3,4-13 C 3 ]-6 [15].…”

Section: Experimental Partmentioning

confidence: 99%

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Structure‐Based Design and Synthesis of the First Weak Non‐Phosphate Inhibitors for IspF, an Enzyme in the Non‐Mevalonate Pathway of Isoprenoid Biosynthesis

Baumgartner

Eberle

Diederich

et al. 2007

Helvetica Chimica Acta

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In this paper, we describe the structure-based design, synthesis, and biological evaluation of cytosine derivatives and analogues that inhibit IspF, an enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. This pathway is responsible for the biosynthesis of the C 5 precursors to isoprenoids, isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2; Scheme 1). The nonmevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non-mevalonate pathway have been identified as attractive new drug targets in the fight against malaria. Based on computer modeling (cf. Figs. 2 and 3), new cytosine derivatives and analogues (Fig. 1) were selected as potential drug-like inhibitors of IspF protein, and synthesized (Schemes 2 -5). Determination of the enzyme activity by 13 C-NMR spectroscopy in the presence of the new ligands showed inhibitory activities for some of the prepared cytosine and pyridine-2,5-diamine derivatives in the upper micromolar range (IC 50 values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the polar diphosphate binding site and the side chain of Asp56', which interacts with the ribose moiety of the substrate and substrate analogues. Furthermore, a new spacious sub-pocket was discovered which accommodates aromatic spacers between cytosine derivatives or analogues (binding to Pocket III ) and rings that occupy the flexible hydrophobic region of Pocket II . The proposed binding mode remains to be further validated by X-ray crystallography.

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Nonmevalonate Terpene Biosynthesis Enzymes as Antiinfective Drug Targets: Substrate Synthesis and High-Throughput Screening Methods

Cited by 52 publications

References 58 publications

Biosynthesis of isoprenoids – studies on the mechanism of 2C‐methyl‐d‐erythritol‐4‐phosphate synthase

Biosynthesis of isoprenoids – studies on the mechanism of 2C‐methyl‐d‐erythritol‐4‐phosphate synthase

Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene

Structure‐Based Design and Synthesis of the First Weak Non‐Phosphate Inhibitors for IspF, an Enzyme in the Non‐Mevalonate Pathway of Isoprenoid Biosynthesis

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