scholarly journals Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate

2002 ◽  
Vol 366 (2) ◽  
pp. 573-583 ◽  
Author(s):  
Jean-François HOEFFLER ◽  
Andréa HEMMERLIN ◽  
Catherine GROSDEMANGE-BILLIARD ◽  
Thomas J. BACH ◽  
Michel ROHMER
Keyword(s):  
Escherichia Coli ◽  
Higher Plants ◽  
Mevalonic Acid ◽  
Cell Suspension Cultures ◽  
Mep Pathway ◽  
Isopentenyl Diphosphate ◽  
Higher Plant ◽  
Methylerythritol Phosphate Pathway ◽  
Dimethylallyl Diphosphate ◽  
Deuterium Retention

In the bacterium Escherichia coli, the mevalonic-acid (MVA)-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is characterized by two branches leading separately to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The signature of this branching is the retention of deuterium in DMAPP and the deuterium loss in IPP after incorporation of 1-[4-2H]deoxy-d-xylulose ([4-2H]DX). Feeding tobacco BY-2 cell-suspension cultures with [4-2H]DX resulted in deuterium retention in the isoprene units derived from DMAPP, as well as from IPP in the plastidial isoprenoids, phytoene and plastoquinone, synthesized via the MEP pathway. This labelling pattern represents direct evidence for the presence of the DMAPP branch of the MEP pathway in a higher plant, and shows that IPP can be synthesized from DMAPP in plant plastids, most probably via a plastidial IPP isomerase.

Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis

2000 ◽  
Vol 353 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Narciso CAMPOS ◽  
Manuel RODRÍGUEZ-CONCEPCIÓN ◽  
Susanna SAURET-GÜETO ◽  
Francesca GALLEGO ◽  
Luisa-María LOIS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Analysis ◽  
Mevalonate Pathway ◽  
Mep Pathway ◽  
Isopentenyl Diphosphate ◽  
Isopentenyl Diphosphate Isomerase ◽  
E Coli ◽  
Dimethylallyl Diphosphate ◽  
And Function ◽  
Synthetic Operon

Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.

scholarly journals Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway.

2001 ◽  
Vol 48 (3) ◽  
pp. 663-672 ◽  
Author(s):  
M Wanke ◽  
K Skorupinska-Tudek ◽  
E Swiezewska
Keyword(s):  
Mevalonate Pathway ◽  
Higher Plants ◽  
Developmental State ◽  
Isoprenoid Biosynthesis ◽  
Mep Pathway ◽  
The Novel ◽  
Dimethylallyl Diphosphate ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Mixed Origin

Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.

scholarly journals Characterization of the Mycobacterium tuberculosis 4-Diphosphocytidyl-2-C-Methyl-d-Erythritol Synthase: Potential for Drug Development

2007 ◽  
Vol 189 (24) ◽  
pp. 8922-8927 ◽  
Author(s):  
Hyungjin Eoh ◽  
Amanda C. Brown ◽  
Lori Buetow ◽  
William N. Hunter ◽  
Tanya Parish ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Divalent Cations ◽  
Mep Pathway ◽  
Isopentenyl Diphosphate ◽  
Dimethylallyl Diphosphate ◽  
Methylerythritol Phosphate ◽  
Ph Range ◽  
Support Activity

ABSTRACT Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-d-erythritol is formed from 2-C-methyl-d-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-d-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated k cat and k cat/Km values were 0.72 min−1 and 12.3 mM−1 min−1 for MEP and 1.0 min−1 and 18.8 mM−1 min−1 for CTP, respectively.

A survey of the presence of glutamate synthase in plant cell suspension cultures

1976 ◽  
Vol 54 (24) ◽  
pp. 2924-2927 ◽  
Author(s):  
Donald K. Dougall ◽  
Jeff Bloch
Keyword(s):  
Cell Suspension ◽  
Higher Plants ◽  
Cell Suspension Cultures ◽  
Glutamate Synthase ◽  
Pyridine Nucleotide ◽  
Suspension Cultures ◽  
Higher Plant ◽  
Plant Cell Suspension Cultures ◽  
Plant Cell Suspension ◽  
Glutamic Dehydrogenase

Evidence was sought for the presence of glutamate synthase (EC 2.6.1.53) in extracts from suspension cultures of six higher plant species not previously examined. The level of glutamate synthase measured was above the level of glutamic dehydrogenase (EC 1.4.1.2, 1.4.1.4) in extracts of soybean, parsley, okra, and cotton. Glutamate synthase was detectable but less than glutamic dehydrogenase in extracts of sugarcane. Glutamate synthase was not detected in extracts of peanut. Evidence for two glutamate synthases, each specific for one pyridine nucleotide, was obtained with cultures of carrot. Glutamate synthase has now been detected in eight and possibly nine species representing four and possibly five families of higher plants.

Herbicidal Inhibition of Carotenogenesis Detected by HPLC

1990 ◽  
Vol 45 (5) ◽  
pp. 492-497 ◽  
Author(s):  
Paul Barry ◽  
Ken E. Pallett
Keyword(s):  
Cell Suspension ◽  
Hplc Analysis ◽  
Biosynthetic Pathway ◽  
Higher Plants ◽  
Cell Suspension Cultures ◽  
Carotenoid Biosynthesis ◽  
Higher Plant ◽  
Target Sites ◽  
Carrot Cell ◽  
Carrot Cell Suspension

The target sites of three herbicides which inhibit carotenoid biosynthesis have been characterized using HPLC analysis of pigment extracts from two higher plant systems, carrot cell suspension cultures and barley seedlings. Diflufenican causes an accumulation of phytoene and phytofluene. Dichlormate causes accumulation of phytoene, phytofluene, ξ-carotene, neurosporene and β-zeacarotene. Amitrole causes accumulation of phytoene, phytofluene, β- γ- and δ-carotenes and lycopene. Significant differences in the geometric and hydroxylated natures of the accumulated precursors occurred between the carrot cell and dark- and light-grown barley. These differences are discussed with respect to both the target sites of the three carotenogenic herbicides and the biosynthetic pathway leading to carotenoid biosynthesis in higher plants.

Sulphate Assimilation in Higher Plants: A Thioredoxin-Dependent PAPS-Reductase from Spinach Leaves

1989 ◽  
Vol 44 (5-6) ◽  
pp. 504-508 ◽  
Author(s):  
J. D. Schwenn
Keyword(s):  
Escherichia Coli ◽  
Higher Plants ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Size Exclusion ◽  
Partial Purification ◽  
Higher Plant ◽  
Inorganic Sulphate ◽  
Sulphate Assimilation ◽  
Spinach Leaves

Abstract Higher plant leaf protein was investigated for the enzyme activity catalyzing a thioredoxin-dependent reduction of 3'-phosphoadenylylsulphate (PAPS) to sulphite. The enzyme became detectable when heterologous thioredoxin from Escherichia coli was used substituting for the hitherto unidentified plant thioredoxin. The enzyme's cross-reactivity with heterologous thioredo­ xin enabled the partial purification and brief characterization. The molecular weight of the en­ zyme as estimated by HPLC size exclusion and gel filtration was 68-72 k. The protein reduced PAPS only when thioredoxin was present as cosubstrate. The function of this enzyme in the assimilation of inorganic sulphate by higher plants is discussed in comparison to the function of the respective enzymes from Escherichia coli and Saccharom yces cerevisiae.

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