Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in <i>Escherichia coli</i>

Rodríguez‐Concepción, Manuel; Campos, Narciso; Lois, L. María; Maldonado, Carlos; Hoeffler, Jean-François; Grosdemange-Billiard, Catherine; Rohmer, Michel; Boronat, Albert

doi:10.1016/s0014-5793(00)01552-0

Cited by 101 publications

(92 citation statements)

References 36 publications

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“…Accordingly, this protein can be identified as the responsible agent for the branching in the nonmevalonate pathway, which had been postulated earlier to account for the anomalous results observed in feeding experiments with deuterated substrates both with E. coli (46,47) and Eucalyptus globulus (48). Independent genetic evidence for the existence of such a branching has been provided for the machinery of E. coli (43). Elucidation of the role of the protein encoded by the ispH gene fills the last gap in the sequence of enzymatic steps of the pathway that leads from pyruvate and D-glyceraldehyde 3-phosphate to IPP and DMAPP.…”

Section: Discussionmentioning

confidence: 77%

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Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein

Rohdich

Hecht²,

Gärtner³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

315

231

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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-Dxylulose 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 C5]1-deoxy-D-xylulose into a 5:1 mixture of [U-13 C5]isopentenyl diphosphate and [U-13 C5]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.T erpenes are one of the largest groups of natural products comprising numerous medically relevant compounds (e.g., vitamins, hormones, and antitumor agents such as Taxol) (1). Bloch, Lynen, Cornforth, and their coworkers showed that the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are biosynthesized by means of the mevalonate pathway in yeasts and animals (for review see refs. 2-5). These studies served as the basis for the development of metabolic inhibitors that are widely used for the treatment of hypercholesterolemia.For a period of several decades, these milestone achievements completely eclipsed the existence of a second terpenoid pathway for the biosynthesis of IPP and DMAPP. Recently, however, knowledge on that pathway has been unfolding rapidly on the basis of seminal discoveries by the research groups of . Briefly, the mevalonate independent pathway starts from 1-deoxy-D-xylulose 5-phosphate, which is assembled from pyruvate and D-glyceraldehyde 3-phosphate (13, 14) and was already known to serve as a biosynthetic precursor of vitamins B 1 (thiamine) and B 6 (pyridoxal) (Fig. 1) (15-17). 1-Deoxy-D-xylulose 5-phosphate is converted into 2C-methyl-D-erythritol 4-phosphate, the first committed intermediate of the nonmevalonate pathway, by isomerization followed by a two-electron reduction step catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase specified by the ispC gene (formerly designated yaeM and then dxr) (18) (Fig. 1).In the next step, 2C-methyl-D-erythritol 4-phosphate is converted into the 2C-methyl-D-erythritol 2,4-cyclodiphosphate by the sequential action of three enzymes specified by the ispD, ispE, and ispF genes (19-24). The last known step of the sequence, the reductive transformation of the cyclic diphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate has been identified recently in work with a recombinant Escherichia coli strain engineered for hyperexpression of the ispG gene (previously designated gcpE) (25). Comparative genomics suggest that the protein specified by the lytB gene is involved in the conversion of ...

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Section: Discussionmentioning

confidence: 77%

“…In the present case sequential formation of the two compounds seems unlikely for a variety of reasons. (i) The IspH protein does not display isomerase activity (27,43). (ii) The idi gene of E. coli encodes an IPP isomerase (44).…”

Section: Discussionmentioning

confidence: 99%

Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein

Rohdich

Hecht²,

Gärtner³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

315

231

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“…Escherichia coli strains were grown in LB broth or plates. When indicated, the growth medium was supplemented with different concentrations of fosmidomycin (Molecular Probes) or 1 mM MVA prepared as described (27). Unless stated otherwise, chemicals and reagents were obtained from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria

Sangari

Pérez-Gil

Carretero‐Paulet

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Isoprenoids are a large family of compounds with essential functions in all domains of life. Most eubacteria synthesize their isoprenoids using the methylerythritol 4-phosphate (MEP) pathway, whereas a minority uses the unrelated mevalonate pathway and only a few have both. Interestingly, Brucella abortus and some other bacteria that only use the MEP pathway lack deoxyxylulose 5-phosphate (DXP) reductoisomerase (DXR), the enzyme catalyzing the NADPH-dependent production of MEP from DXP in the first committed step of the pathway. Fosmidomycin, a specific competitive inhibitor of DXR, inhibited growth of B. abortus cells expressing the Escherichia coli GlpT transporter (required for fosmidomycin uptake), confirming that a DXR-like (DRL) activity exists in these bacteria. The B. abortus DRL protein was found to belong to a family of uncharacterized proteins similar to homoserine dehydrogenase. Subsequent experiments confirmed that DRL and DXR catalyze the same biochemical reaction. DRL homologues shown to complement a DXR-deficient E. coli strain grouped within the same phylogenetic clade. The scattered taxonomic distribution of sequences from the DRL clade and the occurrence of several paralogues in some bacterial strains might be the result of lateral gene transfer and lineage-specific gene duplications and/or losses, similar to that described for typical mevalonate and MEP pathway genes. These results reveal the existence of a novel class of oxidoreductases catalyzing the conversion of DXP into MEP in prokaryotic cells, underscoring the biochemical and genetic plasticity achieved by bacteria to synthesize essential compounds such as isoprenoids.I soprenoids, one of the largest groups of natural compounds, have a variety of roles in respiration, photosynthesis, membrane structure, allelochemical interactions, and growth regulation (1-3). All free-living organisms synthesize isoprenoids from the five carbon precursors isopentenyl diphosphate (IPP) and its double-bond isomer dimethylallyl diphosphate (DMAPP). For decades it was believed that IPP was exclusively synthesized from acetyl coenzyme A by the mevalonate (MVA) pathway and then converted into DMAPP by a IPP/DMAPP isomerase (IDI) enzyme (4). However, in the early nineties of the last century it was discovered that IPP and DMAPP could be formed simultaneously from pyruvate and glyceraldehyde 3-phosphate by an alternative route currently known as the methylerythritol 4-phosphate (MEP) pathway (5, 6). It is now well established that most organisms only use one of the two pathways for isoprenoid biosynthesis. Thus, archaea (archaebacteria), fungi, and animals synthesize IPP from MVA, whereas most bacteria (eubacteria) only use the MEP pathway for the production of isoprenoid precursors. Plants employ both pathways, but in different cell compartments: The MVA pathway synthesizes cytosolic isoprenoid precursors whereas the MEP pathway is located in plastids (7).Because the MEP pathway is absent from animals (including humans) but it is essential in a large number ...

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“…7 In the second step, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR, also referred to as IspC; EC 1.1.1.267) catalyzes the NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate (DXP) to form 2-C-methyl-D-erythritol 4-phosphate (MEP), a reaction that also requires the presence of a divalent cation such as Mg 2+ , Co 2+ or Mn 2+ . 8 Knockouts of the dxr gene in Escherichia coli are lethal, 9 and the essentiality of the M. tuberculosis dxr gene for growth in vitro has also been demonstrated. 10 The natural antibiotic fosmidomycin ( Figure 1) is a known inhibitor of the non-mevalonate pathway in plants and bacteria.…”

Section: Introductionmentioning

confidence: 99%

Design, Synthesis, and X-ray Crystallographic Studies of α-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase

et al. 2011

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The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. α-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin–DXR complex. Our best inhibitor has an IC50 = 0.15 μM on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 μg/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

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Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli

Cited by 101 publications

References 36 publications

Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein

Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein

A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria

Design, Synthesis, and X-ray Crystallographic Studies of α-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase

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