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Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition.

The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.

Animals↗

Overexpression of the Saccharomyces cerevisiae RER2 gene in Trichoderma reesei affects dolichol dependent enzymes and protein glycosylation.

Protein secretion in Trichoderma reesei could be stimulated by overexpression of the yeast Saccharomyces cerevisiae DPM1 gene encoding dolichyl phosphate mannose synthase (DPMS) a key enzyme in the O-glycosylation pathway. The secreted proteins were glycosylated to the wild type level. On the other hand, the elevated concentration of GDP-mannose, a direct substrate for DPMS, resulting from overexpression in T. reesei of the mpg1 gene coding for guanyltransferase, did not affect secretion of proteins but did affect the degree of their O- and N-glycosylation. In this paper, we examined the effects of dolichol, an indispensable carrier of sugar residues in protein glycosylation, on the synthesis of glycosylated proteins. An increase in dolichol synthesis was obtained by overexpression of the yeast gene encoding cis-prenyltransferase, the first enzyme of the mevalonate pathway committed to dolichol biosynthesis. We observed that, an increased concentration of dolichol resulted in an increased expression of the dpm1 gene and DPMS activity and in overglycosylation of secreted proteins.

Chitin↗

Bisphosphonates--mechanisms of action in multiple myeloma.

Bisphosphonates are a class of anti-resorptive drugs, which are effective in the treatment of osteoclast-mediated bone disease, including the osteolytic bone disease. which is a major clinical feature of patients with multiple myeloma. Recently, increases in survival following treatment with pamidronate have been observed in some patients with multiple myeloma, raising the possibility that bisphosphonates may also have an anti-tumour effect. We have demonstrated that bisphosphonates can have an anti-tumour effect in human myeloma cell in vitro, and that these anti-tumour effects induced by potent nitrogen-containing bisphosphonates are a result of inhibition of enzymes of the mevalonate pathway. However, we and others have been unable to demonstrate an anti-tumour effect of the potent bisphosphonate ibandronate in vivo, using murine models of multiple myeloma. It is therefore likely that only by studying patients receiving bisphosphonates will we be able to determine whether these compounds have a clinically important anti-tumour effect.

Animals↗

Biosynthesis of domoic acid by the diatom Pseudo-nitzschia multiseries.

The biosynthesis of the neurotoxin domoic acid (DA) in the diatom Pseudo-nitzschia multiseries was investigated using 13C- and 14C-labelled precursors. The labelling pattern determined by NMR spectroscopy following incorporation of [1,2-13C2]-acetate showed enrichment of every carbon of DA. The enrichment levels were consistent with a biosynthetic pathway involving two different intermediate precursor units. Addition of labelled acetate either early or late during exponential growth gave similar patterns and levels of incorporation. Analysis of the labelling pattern indicated that DA is biosynthesised by condensation of an isoprenoid intermediate with another intermediate derived from the tricarboxylic acid (TCA) cycle. The absence of deuterium at C2 in DA following incorporation of [2-13C, 2H3]-acetate is consistent with alpha-ketoglutarate or a derivative as the TCA cycle-derived intermediate. The different incorporation efficiencies of acetate into the putative precursor intermediates suggest that either each unit is biosynthesized in a different part of the diatom cell, or that the isoprene chain is not assembled by the usual acetate-mevalonate pathway. The latter proposal is supported by the complete absence of deuterium retention in the isoprenoid-derived portion following incorporation of [2-13C, 2H3]-acetate.

Acetates↗

Cholesterol inhibition, cancer, and chemotherapy.

An important feature of malignant transformation is loss of the cholesterol feedback inhibition mechanism that regulates cholesterol synthesis. Cancer cells seem to require an increase in the concentrations of cholesterol and of cholesterol precursors. Therefore, a reasonable assumption is that prevention of tumour-cell growth can be achieved by restricting either cholesterol availability or cholesterol synthesis. In-vivo and cell-culture experiments have shown that lowering the plasma cholesterol concentration or intervening in the mevalonate pathway with 3-hydroxy-3-methylglutaryl (HMG) CoA reductase inhibitors decreases tumour growth. Currently prescribed doses of HMG-CoA reductase inhibitors given orally or continuously by an implantable infusion pump could achieve tumour therapeutic tissue concentrations of these agents. My hypothesis is that cholesterol inhibition can inhibit tumour cell growth, can act as an adjuvant to cancer chemotherapy, and, possibly, can prevent carcinogenesis.

Adjuvants, Pharmaceutic↗

Mevalonate-dependent inhibition of transendothelial migration and chemotaxis of human peripheral blood neutrophils by pravastatin.

Pravastatin, a hydrophilic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, has been reported to beneficially affect atherogenesis, plaque stability, and transient myocardial ischemia in significant coronary artery disease by influencing lipid metabolism and by intracellular signaling via mevalonate pathway products other than cholesterol. Leukocytes are implicated to play a pathophysiological role in these events. We were interested in finding out whether pravastatin could affect transendothelial migration (TEM), chemotaxis, and respiratory burst activity of the neutrophil ex vivo. In addition, effects on monocyte and T-lymphocyte chemotaxis were tested. For TEM assays, monolayers of human umbilical vein endothelial cells (HUVECs) were grown to confluence on polycarbonate filters bearing 5-microns pores in Transwell (Costar) culture plate inserts. Chemotaxis experiments were performed using modified Boyden chambers with cellulose nitrate micropore filters. Respiratory burst activity was measured fluorometrically. Treatment of neutrophils and monocytes with pravastatin at 2 to 200 mumol/L and 10 to 1000 mumol/L, respectively, significantly decreased chemotaxis triggered by fMet-Leu-Phe. This effect was abolished in the presence of mevalonic acid (500 mumol/L); no effect of pravastatin was seen on T-lymphocyte chemotaxis triggered by interleukin-8. Preincubation of neutrophils with pravastatin (200 mumol/L) also resulted in a significant reduction in the number of neutrophils that transmigrated a tumor necrosis factor-stimulated or lipopolysaccharide-stimulated HUVEC monolayer. At none of the concentrations tested (2 pmol/L to 200 mumol/L) did pravastatin affect neutrophil respiratory burst activity. We conclude that pravastatin may alter monocyte chemotaxis and neutrophil-endothelial interactions in migratory responses at concentrations obtained in vivo with cholesterol-lowering doses.

Cell Movement↗

Genetic evaluation of physiological functions of thiolase isoenzymes in the n-alkalane-assimilating yeast Candida tropicalis.

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1a delta/t1bdelta null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3a delta/t3b delta null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C10 to C13). All thiolase activities in this yeast disappeared with the ct-t1a delta/t1bdelta and ct-t3a delta/t3bdelta null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bdelta null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal beta-oxidation. In addition to its essential contribution to the beta-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the beta-oxidation of a C4 substrate butyrate.

Acetyl-CoA C-Acetyltransferase↗

Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives.

The mevalonate pathway is tightly linked to cell proliferation. The aim of the present study is to determine the relationship between the inhibition of this pathway by lovastatin and the cell cycle. HL-60 and MOLT-4 human cell lines were cultured in a cholesterol-free medium and treated with increasing concentrations of lovastatin, and their effects on cell proliferation and the cell cycle were analyzed. Lovastatin was much more efficient in inhibiting cholesterol biosynthesis than protein prenylation. As a result of this, lovastatin blocked cell proliferation at any concentration used, but its effects on cell cycle distribution varied. At relatively low lovastatin concentrations (less than 10 microM), cells accumulated preferentially in G(2) phase, an effect which was both prevented and reversed by low-density lipoprotein cholesterol. At higher concentrations (50 microM), the cell cycle was also arrested at G(1) phase. In cells treated with lovastatin, those arrested at G(1) progressed through S upon mevalonate provision, whereas cholesterol supply allowed cells arrested at G(2) to traverse M phase. These results demonstrate the distinct roles of mevalonate, or its non-sterol derivatives, and cholesterol in cell cycle progression, both being required for normal cell cycling.

Apoptosis↗

A novel biosynthetic pathway providing precursors for fatty acid biosynthesis and secondary metabolite formation in myxobacteria.

Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in the esg mutant. Feeding experiments using (13)C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employing Myxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well.

Bacterial Proteins↗

Regulatory aspects of coenzyme Q metabolism.

A number of factors are involved in the regulation of the amount and distribution of coenzyme Q in cells and tissues. These factors modify preferentially the biosynthetic mechanism in order to keep up an optimal tissue concentration of the lipid. The amount of substrate provided by the mevalonate pathway is able to both up- and down-regulate the velocity of synthesis. At the translation level, regulation occurs by receptor-mediated ligand binding and appears most clearly upon treatment with hormones and peroxisomal inducers. There are a number of pathophysiological conditions when these mechanisms of regulation are modified and explain the decreased coenzyme Q tissue concentrations. It is of considerable interest to establish appropriate physiological, hormonal and drug-mediated conditions in order to counteract disturbed cellular functions caused by coenzyme Q deficiency

Aging↗

The bisphosphonate zoledronic acid impairs Ras membrane [correction of impairs membrane] localisation and induces cytochrome c release in breast cancer cells.

Bisphosphonates are well established in the management of cancer-induced bone disease. Recent studies have indicated that these compounds have direct inhibitory effects on cultured human breast cancer cells. Nitrogen-containing bisphosphonates including zoledronic acid have been shown to induce apoptosis associated with PARP cleavage and DNA fragmentation. The aim of this study was to identify the signalling pathways involved. Forced expression of the anti-apoptotic protein bcl-2 attenuated bisphosphonate-induced loss of cell viability and induction of DNA fragmentation in MDA-MB-231 cells. Zoledronic acid-mediated apoptosis was associated with a time and dose-related release of mitochondrial cytochrome c into the cytosol in two cell lines. Rescue of cells by preincubation with a caspase-3 selective inhibitor and demonstration of pro-caspase-3 cleavage products by immunoblotting suggests that at least one of the caspases activated in response to zoledronic acid treatment is caspase-3. In both MDA-MB-231 and MCF-7 breast cancer cells, zoledronic acid impaired membrane localisation of Ras indicating reduced prenylation of this protein. These observations demonstrate that zoledronic acid-mediated apoptosis is associated with cytochrome c release and consequent caspase activation. This process may be initiated by inhibition of the enzymes in the mevalonate pathway leading to impaired prenylation of key intracellular proteins including Ras.

Apoptosis↗

Early embryonic lethality caused by targeted disruption of the 3-hydroxy-3-methylglutaryl-CoA reductase gene.

The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/-) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/- embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/- mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/- and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.

Alleles↗

HMG-CoA reductase inhibitors suppress macrophage growth induced by oxidized low density lipoprotein.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors ameliorate atherosclerotic diseases in several models of vascular disease. This is largely due to their ability to reduce plasma cholesterol levels in vivo. Proliferation of cellular components is one of the major events in the development and progression of atherosclerotic lesions. We recently demonstrated that oxidized low density lipoprotein (Ox-LDL), a likely atherogenic lipoprotein present in vivo, is capable of inducing macrophage growth in vitro. In the present study, we investigated the effect of HMG-CoA reductase inhibitors, simvastatin and pravastatin, on Ox-LDL-induced macrophage growth. Our results demonstrated that these inhibitors effectively suppressed Ox-LDL-induced macrophage growth with concentrations required for 50% inhibition by simvastatin and pravastatin being 0.1 and 80 microM, respectively, and that this inhibitory effect was reversed by mevalonate but not by squalene. Under these conditions, simvastatin did not affect the endocytic degradation of Ox-LDL, nor subsequent accumulation of intracellular cholesteryl esters. Our results suggest that a non-cholesterol metabolites(s) of mevalonate pathway may play an important role in Ox-LDL-induced macrophage growth. Since it is well known that macrophage-derived foam cells are the key cellular element in the early stage of atherosclerosis, a significant inhibition of Ox-LDL-induced macrophage growth by HMG-CoA reductase inhibitors in vitro, particularly simvastatin, may also explain, at least in part, their anti-atherogenic action in vivo.

Animals↗

Bisphosphonates: from the laboratory to the clinic and back again.

Bisphosphonates (BPs) used as inhibitors of bone resorption all contain two phosphonate groups attached to a single carbon atom, forming a "P-C-P" structure. The bisphosphonates are therefore stable analogues of naturally occuring pyrophosphate-containing compounds, which now helps to explain their intracellular as well as their extracellular modes of action. Bisphosphonates adsorb to bone mineral and inhibit bone resorption. The mode of action of bisphosphonates was originally ascribed to physico-chemical effects on hydroxyapatite crystals, but it has gradually become clear that cellular effects must also be involved. The marked structure-activity relationships observed among more complex compounds indicate that the pharmacophore required for maximal activity not only depends upon the bisphosphonate moiety but also on key features, e.g., nitrogen substitution in alkyl or heterocyclic side chains. Several bisphosphonates (e.g., etidronate, clodronate, pamidronate, alendronate, tiludronate, risedronate, and ibandronate) are established as effective treatments in clinical disorders such as Paget's disease of bone, myeloma, and bone metastases. Bisphosphonates are also now well established as successful antiresorptive agents for the prevention and treatment of osteoporosis. In particular, etidronate and alendronate are approved as therapies in many countries, and both can increase bone mass and produce a reduction in fracture rates to approximately half of control rates at the spine, hip, and other sites in postmenopausal women. In addition to inhibition of osteoclasts, the ability of bisphosphonates to reduce the activation frequency and birth rates of new bone remodeling units, and possibly to enhance osteon mineralisation, may also contribute to the reduction in fractures. The clinical pharmacology of bisphosphonates is characterized by low intestinal absorption, but highly selective localization and retention in bone. Significant side effects are minimal. Current issues with bisphosphonates include the introduction of new compounds, the choice of therapeutic regimen (e.g., the use of intermittent dosing rather than continuous), intravenous vs. oral therapy, the optimal duration of therapy, the combination with other drugs, and extension of their use to other conditions, including steroid-associated osteoporosis, male osteoporosis, arthritis, and osteopenic disorders in childhood. Bisphosphonates inhibit bone resorption by being selectively taken up and adsorbed to mineral surfaces in bone, where they interfere with the action of osteoclasts. It is likely that bisphosphonates are internalized by osteoclasts and interfere with specific biochemical processes and induce apoptosis. The molecular mechanisms by which these effects are brought about are becoming clearer. Recent studies show that bisphosphonates can be classified into at least two groups with different modes of action. Bisphosphonates that closely resemble pyrophosphate (such as clodronate and etidronate) can be metabolically incorporated into nonhydrolysable analogues of ATP that may inhibit ATP-dependent intracellular enzymes. The more potent, nitrogen-containing bisphosphonates (such as pamidronate, alendronate, risedronate, and ibandronate) are not metabolized in this way but can inhibit enzymes of the mevalonate pathway, thereby preventing the biosynthesis of isoprenoid compounds that are essential for the posttranslational modification of small GTPases. The inhibition of protein prenylation and the disruption of the function of these key regulatory proteins explains the loss of osteoclast activity and induction of apoptosis. These different modes of action might account for subtle differences between compounds in terms of their clinical effects. In conclusion, bisphosphonates are now established as an important class of drugs for the treatment of bone diseases, and their mode of action is being unravelled. As a result, their full therapeutic potential is gradual

Diphosphonates↗

HMG-CoA reductase inhibitor simvastatin mitigates VEGF-induced "inside-out" signaling to extracellular matrix by preventing RhoA activation.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors exert modulatory effects on a number of cell signaling cascades by preventing the synthesis of various isoprenoids derived from the mevalonate pathway. In the present study, we describe a novel pleiotropic effect of HMG-CoA reductase inhibitors, also commonly known as statins, on vascular endothelial growth factor (VEGF)-induced type IV collagen accumulation. VEGF is an angiogenic polypeptide that is also known to play a central role in endothelial cell permeability and differentiation. Recently, VEGF has also been implicated in promoting extracellular matrix (ECM) accumulation, although the precise signaling mechanism that mediates VEGF-induced ECM expansion remains poorly characterized. Elucidation of the mechanisms through which VEGF exerts its effect on ECM is clearly a prerequisite for both understanding the complex biology of this molecule as well as targeting VEGF in several pathological processes. To this end, this study explored the underlying molecular mechanisms mediating VEGF-induced ECM expansion in mesangial cells. Our findings show that VEGF stimulation elicits a robust increase in ECM accumulation that involves RhoA activation, an intact actin cytoskeleton, and beta(1)- integrin activation. Our data also indicate that simvastatin, via mevalonate depletion, reverses VEGF-induced ECM accumulation by preventing RhoA activation.

Actin Cytoskeleton↗

The bisphosphonate acute phase response: rapid and copious production of proinflammatory cytokines by peripheral blood gd T cells in response to aminobisphosphonates is inhibited by statins.

The bisphosphonates are a novel class of drug that have been registered for various clinical applications worldwide. Bisphosphonates, and in particular the aminobisphosphonates (nBPs), are known to have a number of side-effects including a rise in body temperature and accompanying flu-like symptoms that resemble a typical acute phase response. The mechanism for this response has been partially elucidated and appears to be associated with the release of tumour necrosis factor (TNF)alpha and interleukin (IL)6, although the effector cells that release these cytokines and the mechanism of action remain enigmatic. Here, we show that the nBP-induced acute phase response differs from the typical acute phase response in that CD14+ cells such as monocytes and macrophages are not the primary cytokine producing cells. We show that by inhibiting the mevalonate pathway, nBPs induce rapid and copious production of TNFalpha and IL6 by peripheral blood gammadelta T cells. Prior treatment with statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, blocks nBP-induced production of these proinflammatory cytokines by gammadelta T cells and may offer a means of avoiding the associated acute phase response. In addition, our findings provide a further mechanism for the anti-inflammatory effects attributed to inhibitors of HMG CoA reductase.

Acute-Phase Reaction↗

Identification of an Archaeal type II isopentenyl diphosphate isomerase in methanothermobacter thermautotrophicus.

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70 degrees C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K(m), 64 microM; specific activity, 0.476 micromol mg(-1) min(-1); and k(cat), 1.6 s(-1).

Amino Acid Sequence↗

Functional involvement of a deoxy-D-xylulose 5-phosphate reductoisomerase gene harboring locus of Synechococcus leopoliensis in isoprenoid biosynthesis.

The present work aimed to proof the functionality of the non-mevalonate pathway in cyanobacteria. It was intended to isolate the 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase gene (dxr), as this gene encodes the enzyme which catalyzes a pathway-specific, indicative step of this pathway. For this purpose, a segment of dxr was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with the PCR segment led to the identification of a 26. 5 kbp locus on which a dxr homologous gene and two adjacent open reading frames organized in one operon were localized by DNA sequencing. The functionality of the gene was demonstrated expressing the gene in Escherichia coli and using the purified gene product in a photometrical NADPH dependent test based on the substrate DXP generating system. While the content of one of the central intermediates of the isoprenoid biosynthesis (dimethylallyl diphosphate=DMADP) was significantly (P</=0.001) increased in E. coli cells overexpressing the DXP synthase gene (dxs) of S. leopoliensis, overexpression of dxr does not lead to an elevated DMADP level. Since even in strains harboring an expression fusion of dxs the additional overexpression of dxr does not influence the DMADP content, it is concluded that Dxs but not Dxr catalyzes a rate limiting step of the non-mevalonate isoprenoid biosynthesis.

Aldose-Ketose Isomerases↗