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PubMed1998

On the absence of the glyceraldehyde 3-phosphate/pyruvate pathway for isoprenoid biosynthesis in fungi and yeasts.

The biosynthesis of isopentenyl diphosphate, the central intermediate of isoprenoid formation, was investigated in the fungus Aschersonia aleyrodis and the yeast Rhodotorula glutinis. The incorporation of 13C-labeled glucose or acetate into their isoprenoids showed that ergosterol in both micro-organisms, ubiquinone in R. glutinis and dihydro-ubiquinone, beta-carotene and triterpenes of the hopane series in A. aleyrodis were synthesized via the mevalonate pathway. No evidence for the presence of the alternative mevalonate-independent glyceraldehyde 3-phosphate/pyruvate pathway was found.

Acetates↗
PubMed1998

A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis.

Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli.

Aldose-Ketose Isomerases↗
PubMed2003

Biosynthesis of isoprenoids: crystal structure of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase.

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.

Adenosine Triphosphate↗
PubMed2006

The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding.

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of approximately 200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis.

Crystallization↗
PubMedDate not supplied

[Isoprenoid pigments in representatives of the family Microbacteriaceae].

By using fosmidomycin and mevinolin (inhibitors of the synthesis of isoprenoid pigments), spectrophotometry, and mass spectrometry, the presence of isoprenoid pigments is shown in 71 of the 78 strains under study. All of these strains belong to 11 genera of the family Microbacteriaceae. Yellow, orange, and red pigments are found to have absorption spectra typical of C40-carotenoids. Eight out of the sixteen strains of the genus Microbacterium are able to synthesize neurosporene, a precursor of lycopene and beta-carotene. The biosynthesis of carotenoids in some representatives of the genera Agromyces, Leifsonia, and Microbacterium is induced by light. Inhibition of the biosynthesis of isoprenoid pigments by fosmidomycin suggests that they are synthesized via the nonmevalonate pathway. Twelve strains are found to exhibit both the nonmevalonate and mevalonate pathways of isoprenoid synthesis. These data, together with the difference in the inhibitory concentration of fosmidomycin, can be used for differentiating various taxa within the family Microbacteriaceae.

Actinomycetales↗
PubMed2005

Kinetic characterization of Synechocystis sp. PCC6803 1-deoxy-D-xylulose 5-phosphate reductoisomerase mutants.

The methylerythritol phosphate pathway to isoprenoids has been firmly established as an alternate to the mevalonate pathway in many bacteria, plants, algae, and the malaria parasite Plasmodium falciparum. The second enzyme in this pathway, deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; E.C. 1.1.1.267), has been the focus of many investigations since it was found to be the target of the antibacterial and antimalarial compound, fosmidomycin. Several x-ray crystal structures of the Escherichia coli and Zymomonas mobilis DXR enzymes have provided important structural information about the residues potentially involved in substrate binding and catalysis. Site-directed mutagenesis studies can be used to complement the structural studies, providing kinetic data for specific changes of active site residues. Active site mutants were prepared of the recombinant Synechocystis sp. PCC6803 DXR, targeting residues D152, S153, E154, H155, M206, and E223. Alteration of the three acidic residues had major effects on catalysis, changes to S153 and M206 had variable effects on binding and catalysis, and a H155A mutation had only minimal effects on the kinetic parameters.

Aldose-Ketose Isomerases↗
PubMed1997

Light modulates the spatial patterns of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in Arabidopsis thaliana.

Although the coordinated biosynthesis of isoprenoid compounds is thought to be essential to the normal processes of plant growth and development, the mechanisms that regulate the mevalonate pathway in plants are not well understood. As the first committed step in the pathway, the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonic acid by HMG CoA reductase and the regulation of the genes encoding this enzyme have been implicated in the network that controls isoprenoid biosynthesis in higher plants. Using histochemical staining for beta-glucuronidase, as well as conventional RNA hybridization analysis, the temporal and spatial regulation of HMG1, one of the genes encoding HMG CoA reductase in the crucifer Arabidopsis thaliana, has been characterized. Furthermore, the HMG1 promoter is shown to be differentially responsive to illumination in different organs, and promoter activation by light deprivation is confined primarily to immature leaves. In contrast, expression of the HMG1 gene in roots is confined to the elongation zone and is not responsive to illumination. Light-mediated regulation of HMG1 expression is shown to be an organ-autonomous response that depends on direct illumination, and environmental cues regarding light do not appear to be exchanged between different organs in Arabidopsis. These studies reveal several new features of HMG1 regulation, and indicate that the high levels of HMG CoA reductase expression detected in immature leaves may be primarily attributed to the dark-induced expression of HMG1, and that HMG1 is expressed at low levels throughout the plant in response to light. Thus, environmental cues interact with the developmental program to define the pattern of HMG1 gene expression in Arabidopsis.

Arabidopsis↗
PubMed1985

Studies on the diurnal rhythm of mevalonate metabolism by sterol and nonsterol pathways and of mevalonate-activating enzymes.

Both in vivo and in vitro incorporation of mevalonic acid into nonsaponifiable lipids by 17-day-old chick liver and kidney did not show diurnal rhythm. Using 14CO2 production from MVA as an index of the shunt pathway not leading to sterols, we have demonstrated for the first time that there is no diurnal rhythm in this pathway. No significant differences were found in the specific activities of mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase from chick liver and kidney throughout a period of 24 hr, using [1-14C]mevalonate as substrate. The absence of diurnal rhythm in the decarboxylase activity was corroborated by further experiments carried out using [2-14C]mevalonate-5-pyrophosphate as specific substrate of this enzyme.

Animals↗
PubMed2003

Cell-cycle gene expression in lovastatin-induced medulloblastoma apoptosis.

BACKGROUND: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is a key rate-limiting enzyme in the mevalonate pathway, which generates precursors both for cholesterol biosynthesis and for the production of nonsteroidal mevalonate derivatives that are involved in a number of growth-regulatory processes. We have reported that lovastatin, a competitive inhibitor of HMG-CoA reductase, not only inhibits medulloblastoma proliferation in vitro, but also induces near-complete cell death via apoptosis. The mechanism of this phenomenon is unclear. Possible involvement of changes in expression of certain cell-cycle related genes led us to study some of them in more detail. METHODS: Medulloblastoma cell lines were exposed in vitro to lovastatin, and the effects of gene expression changes were studied using RT-PCR, antisense oligonucleotide, DNA electrophoresis and Western blotting analysis. RESULTS: 1) Levels of total Ras gene mRNA and individual Ras gene mRNA are stable in lovastatin treatment in all examined medulloblastoma cell lines. 2) Blocking c-myc gene over-expression does not enhance medulloblastoma cell sensitivity to lovastatin. 3) Following lovastatin treatment, p16 expression exhibits no change, but pronounced increases of p27KIP1 protein are observed in all examined cell lines. Lovastatin induces pronounced increases of p21WAF1 protein only in Daoy and UW228, but not in D283 Med and D341 Med. 4) Following lovastatin treatment, increased p53 protein is detected only in D341 Med, and bax protein is unchanged in all cell lines. CONCLUSIONS: Lovastatin-induced growth inhibition and apoptosis in medulloblastoma are not dependent on the regulation of Ras and c-myc gene expression, but may be mediated by p27KIP1 gene expression. Lovastatin-induced apoptosis in medulloblastoma is probably p53 independent, but p53 and p21WAF1 gene expression may also mediate anti-proliferative effects of lovastatin on specific medulloblastoma cell lines.

Apoptosis↗
PubMed1999

Vgamma9/Vdelta2 T cell activation induced by bacterial low molecular mass compounds depends on the 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid biosynthesis.

Isopentenyl diphosphate (IPP), an important precursor of isoprenoid biosynthesis in prokaryotic and eukaryotic organisms, has been shown to activate Vgamma9/Vdelta2 T cells, the major subset of human gammadelta T cells. The biosynthesis of IPP has been first described as the acetate/mevalonate pathway. Recently, 1-deoxy-D-xylulose 5-phosphate (DOXP) and 2-C-methyl-D-erythritol 4-phosphate have been shown to be key metabolites in the DOXP pathway also leading to the formation of IPP in some eubacteria such as Escherichia coli. Here we report that the low molecular mass fraction of extracts from bacteria using the DOXP pathway induces Vgamma9/Vdelta2 T cell activation, while analogous preparations from bacteria using the classical mevalonate pathway fail to do so. Addition of 1-deoxy-D-xylulose potentiates the ability of E. coli extracts to activate Vgamma9/Vdelta2 T cells. As the amounts of IPP present in the bacterial preparations are not sufficient to induce significant Vgamma9/Vdelta2 T cell activation, our data suggest that compounds other than IPP associated with the DOXP pathway are responsible for Vgamma9/Vdelta2 T cell activation.

Chemical Fractionation↗
PubMed1980

Sex difference in human mevalonate metabolism.

Two pathways of mevalonate metabolism have been demonstrated: the major (sterol) pathway leads to cholesterol synthesis, whereas the second shunts mevalonate away from sterol production and ultimately results in its oxidation to CO2. Previous studies have demonstrated that the female rat metabolizes circulating mevalonate by the shunt pathway at twice the rate of the male, whereas the male rat converts significantly more circulating mevalonate to cholesterol than the female. The present study extends these observations to humans. Six men and five premenopausal women with normal renal function were injected with R,S-[5-14C]mevalonate, and 14CO2 expired in the breath of the subjects was monitored continuously with an ionization chamber. On an average, the female subjects expired 16.5% and the males 9.8% of the injected R-[5-14C]mevalonate (P less than 0.001). No differences were observed in the plasma and erythrocyte [14C]cholesterol levels. These data demonstrate, in human beings, a sex difference in mevalonate metabolism. The overall impact of the greater mevalonate shunt activity on cholesterol balance in women is unknown.

Adult↗
PubMed1999

Impaired regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation in lovastatin-resistant cells.

L-90 cells were selected to grow in the presence of serum lipoproteins and 90 microM lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). L-90 cells massively accumulate HMGR, a result of >10-fold amplification of the gene and 40-fold rise in mRNA, and also overexpress other enzymes of the mevalonate pathway. Western blot and promoter-luciferase analyses indicate that transcriptional regulation of sterol-responsive genes by 25-hydroxycholesterol or mevalonate is normal. Yet, none of these genes is regulated by lipoproteins, a result of severe impairment in the low density lipoprotein receptor pathway. Moreover, L-90 cells do not accelerate the degradation of HMGR or transfected HMGal chimera in response to 25-hydroxycholesterol or mevalonate. This aberrant phenotype persists when cells are grown without lovastatin for up to 37 days. The inability to regulate HMGR degradation is not due to its overproduction since in LP-90 cells, which were selected for lovastatin resistance in lipoprotein-deficient serum, HMGR is overexpressed, yet its turnover is regulated normally. Also, the rapid degradation of transfected alpha subunit of T cell receptor is markedly retarded in L-90 cells. These results show that in addition to gene amplification and overexpression of cholesterogenic enzymes, statin resistance can follow loss of regulated HMGR degradation.

Animals↗
PubMed1999

Induced biosynthesis of insect semiochemicals in plants.

Plants under attack by a herbivore may emit characteristic volatiles that are implicated in the attraction of the natural enemies of the herbivore. The signal cascade between leaf damage and the volatile production is stimulated by high- or low-molecular-weight elicitors from the secretions of the herbivore. Besides compounds from the octadecanoid signalling pathway, several structurally non-related amino acid conjugates such as the bacterial phytotoxin coronatine, the synthetic indanoyl-isoleucine, or amino acid conjugates of linolenic acid likewise induce volatile biosynthesis. Minor changes in the amino acid moiety may result in different volatile profiles (sesqui- and diterpenoids), attributing to the amino acid substructure a specific role for the recognition and the selective induction. Volatile terpenoids (mono- and diterpenoids) are synthesised de novo along the novel deoxy-D-xylulose (DOX) pathway, while the biosynthesis of sesquiterpenes may be fuelled from both the DOX- and the mevalonate pathway. This finding may be of importance for the plant defence in case of introduction of inhibitors together with the salivary secretion of herbivores into the leaf tissue.

Animals↗
PubMed2003

Early steps of deoxyxylulose phosphate pathway in chromoplasts of higher plants.

1-Deoxy-D-xylulose 5-phosphate and 2C-methyl-D-erythritol 4-phosphate have been shown as intermediates of the deoxyxylulose phosphate pathway used for terpenoid biosynthesis in plants and many microorganisms. In plants this non-mevalonate pathway is located in plastids. In order to investigate the formation of five carbon intermediates, chromoplasts from Capsicum annuum and Narcissus pseudonarcissus were incubated with isotope-labeled 1-deoxy-D-xylulose 5-phosphate or 2C-methyl-D-erythritol 4-phosphate. The downstream metabolites were detected and separated by reversed-phase ion-pair radio-HPLC and their structures elucidated by mass spectroscopy. Here we report the isolation and structural identification of 4-diphosphocytidyl-2C-methyl-D-erythritol and 2C-methyl-D-erythritol 2,4-cyclodiphosphate from chromoplasts; the genes of the corresponding enzymes had been previously identified from Escherichia coli and Arabidopsis.

Biotransformation↗
PubMed1994

Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.

Animals↗
PubMed2005

fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis.

Although flavodoxin I is indispensable for Escherichia coli growth, the exact pathway(s) where flavodoxin I is essential has not been identified. We performed transposon mutagenesis of the flavodoxin I gene, fldA, in an E. coli strain that expressed mevalonate pathway enzymes and that had a point mutation in the lytB gene of the MEP pathway resulting in the accumulation of (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP). Disruption of fldA abrogated mevalonate-independent growth and dramatically decreased HMBPP levels. The fldA- mutant grew with mevalonate indicating that the essential role of flavodoxin I under aerobic conditions is in the MEP pathway. Growth was restored by fldA complementation. Since GcpE (which synthesizes HMBPP) and LytB are iron-sulfur enzymes that require a reducing system for their activity, we propose that flavodoxin is essential for GcpE and possibly LytB activity. Thus, the essential role for flavodoxin I in E. coli is in the MEP pathway for isoprenoid biosynthesis.

Amino Acid Substitution↗
PubMed2004

Beta4 integrin is involved in statin-induced endothelial cell death.

HMG-CoA reductase inhibitors (statins) have been shown to inhibit angiogenesis. The molecular mechanism mediating the anti-endothelial activities of statins remains unclear. The present study demonstrated that the antiangiogenic effect of atorvastatin (ATV) was associated with endothelial death. Molecular profiling data identified a 29-fold upregulation of beta4 integrin mRNA. Western blot and flow cytometry confirmed robust increases of total and cell-surface beta4 integrin. Blockage of beta4 integrin activity by antagonizing antibody abrogated ATV-induced endothelial death. The endothelial death and beta4 integrin upregulation by ATV could be reversed by intermediate metabilites of the HMG-CoA reductase pathway mevalonate or GGPP, but not by FPP, suggesting that these effects were results of specific inhibition of the pathway. These data indicate that the HMG-CoA reductase might represent an important survival pathway in angiogenic endothelial cells and thus, a potential target for antiangiogenic therapy.

Apoptosis↗