DOCSLIB.ORG

Mevalonate and Nonmevalonate Pathways for the Biosynthesis of Isoprene Units

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mevalonate and Nonmevalonate Pathways for the Biosynthesis of Isoprene Units Biosci. Biotechnol. Biochem., 66 (8), 1619–1627, 2002 Review Mevalonate and Nonmevalonate Pathways for the Biosynthesis of Isoprene Units Tomohisa KUZUYAMA Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Isoprenoids are synthesized by consecutive condensa- nate pathway in humans. Its speciˆc inhibitors, tions of their ˆve-carbon precursor, isopentenyl pravastatin and related compounds, are widely used diphosphate, to its isomer, dimethylallyl diphosphate. as cholesterol-lowering agents.6) Mevalonate is then Two pathways for these precursors are known. One is phosphorylated twice and decarboxylated to form the mevalonate pathway, which operates in eucaryotes, isopentenyl diphosphate (IPP). IPP is then converted archaebacteria, and cytosols of higher plants. The other to its isomer, dimethylallyl diphosphate (DMAPP), is a recently discovered pathway, the nonmevalonate catalyzed by IPP isomerase. IPP and DMAPP syn- pathway, which is used by many eubacteria, green thesized in the mevalonate pathway are used as basic algae, and chloroplasts of higher plants. To date, ˆve units in the biosynthesis of isoprenoids such as reaction steps in this new pathway and their corre- sterols, carotenoids, and dolichols. sponding enzymes have been identiˆed. EC numbers of Since the discovery of the mevalonate pathway, it these enzymes have been assigned by the Nomenclature was widely accepted that IPP and DMAPP were Committee of the International Union of Biochemistry formed only through this pathway in all living organ- and Molecular Biology (NC-IUBMB) and are available isms. However, several results inconsistent with the at http:WWwww.chem.qmw.ac.ukWiubmbWenzymeWreac- operation of the mevalonate pathway in certain bac- 13 tionWterpWnonMVA.html. teria had been reported. For example, [ C]acetate, a precursor of the mevalonate pathway, was not incor- Key words: isoprenoid; biosynthesis; mevalonate porated into ubiquinone of Escherichia coli 7) or into pathway; nonmevalonate pathway; inhi- pentalenolactone produced by Streptomyces chro- bitor mofuscus (H. Seto, unpublished results).8,9) Feeding experiments with [U-13C]glucose and pentalenolac- In 1956, a newly discovered acetate-replacing tone-producing S. exfoliatus showed labeling pat- factor for Lactobacilli was identiˆed as b-hydroxy- terns inconsistent with the mevalonate pathway.8,9) b-methyl-d-valerolactone by Wolf et al.1) This com- Furthermore, mevinolin, a speciˆc inhibitor of pound also was found to be a precursor of HMG-CoA reductase, did not inhibit the growth of cholesterol.2) In the same year, the chemical structure E. coli.7) These results suggested the existence of an of ``hiochic acid'', a growth factor for Lactobacillus alternative pathway for isoprenoid biosynthesis, homohiochi and L. heterohiochi, also was identiˆed which was not identiˆed for some time. as b-hydroxy-b-methyl-d-valerolactone by Tamura,3) In 1996, Rohmer discovered the ˆrst reaction step whoshowedthatsake (Japanese rice wine) frequently of the alternative, nonmevalonate, pathway for the is spoiled by the bacteria that contain this growth formation of IPP and DMAPP in E. coli.10) My factor. Later, b-hydroxy-b-methyl-d-valerolactone group was encouraged by this pioneering work and and ``hiochic acid'' were renamed mevalonate as began to study the nonmevalonate pathway. This their standard name. review centers mostly on our studies of this alterna- In the 1960s, Bloch and Lynen identiˆed the tive pathway. mevalonate pathway for cholesterol biosynthesis.4,5) In this pathway, three molecules of acetyl-CoA 1. First reaction step, catalyzed by DXP condense successively to form 3-hydroxy-3-methyl- synthase (EC 4.1.3.37) glutaryl-CoA (HMG-CoA) (Fig. 1). This CoA- derivative is reduced to mevalonate by HMG-CoA The initial step in the nonmevalonate pathway is reductase, the rate-limiting enzyme of the mevalo- the formation of 1-deoxy-D-xylulose 5-phosphate To whom correspondence should be addressed. Tel: +81-3-5841-7841; Fax: +81-3-5841-8485; E-mail: kuz＠iam.u-tokyo.ac.jp Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; DX, 1-deoxy-D-xylulose; ME, 2-C-methylerythritol; MEP, 2-C-methyl-D-erythritol 4- phosphate; CDP-ME, 4-(cytidine 5?-diphospho)-2-C-methyl-D-erythritol; CDP-ME2P, 2-phospho-4-(cytidine 5?-diphospho)-2-C-methyl-D- erythritol; MECDP, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; HMBDP, 1-hydroxy-2-methyl-2-(E )-butenyl 4-diphosphate 1620 T. KUZUYAMA and a divalent cation such as Mg2+ or Mn2+ for en- zyme activity. The reaction preceding this condensa- tion had earlier been found by Yokota et al.13,14) They detected the formation of 1-deoxy-D-xylulose (DX) from pyruvate and D-glyceraldehyde by pyruvate de- hydrogenase of E. coli or Bacillus subtilis. We cloned a dxs gene homolog from Streptomyces sp. strain CL190 by the polymerase chain reaction using oligonucleotide primers based on highly con- served amino acid sequences and compared its en- zymatic properties with those of the E. coli DXP syn- thase.15) Although these two enzymes had diŠerent origins, they had almost identical enzymatic proper- ties. Le áon and co-workers isolated albino mutants of Arabidopsis thaliana. The albino phenotype of one of these mutants was restored by the feeding of DX, the free alcohol of DXP.16) This mutant had a muta- tion in the cla1 gene, which coded for an amino acid sequence very similar to that of E. coli DXP syn- thase. The cla1 gene transcript and its protein ac- cumulated in young developing tissues. DXP is a biosynthetic intermediate not only for IPP and DMAPP but also for thiamine and pyri- doxol in E. coli (Fig. 2).17) This ˆnding means that DXP synthase is not the only enzyme speciˆc for the nonmevalonate pathway. 2. Second reaction step, catalyzed by DXP reductoisomerase (EC 1.1.1.267) Rohmer and co-workers found that 2-C- methylerythritol (ME) was incorporated into E. coli cells, which used it as the precursor of the side chain of ubiquinone (Fig. 3(A)).18,19) Because of this ˆnd- ing, the intramolecular rearrangement of DXP had been assumed to yield a hypothetical rearrangement product, 2-C-methylerythrose 4-phosphate, which wasthenconvertedto2-C-methyl-D-erythritol 4- phosphate (MEP) by an unknown reduction process (Fig.3(A)).Forthisreason,itwasassumedthattwo enzymes were involved in the formation of MEP from DXP. Fig. 1. Mevalonate Pathway for the Biosynthesis of IPP and To elucidate the details of this mechanism, we DMAPP. studied the cloning of the gene responsible for MEP In this pathway, three molecules of acetyl-CoA condense suc- cessively to form HMG-CoA. This CoA-derivative is reduced to synthesis. We used the strategy of screening for E. mevalonate by HMG-CoA reductase, the rate-limiting enzyme coli mutants with a metabolic block(s) between DXP of this pathway. Mevalonate is then phosphorylated twice and and MEP. Such mutants would need MEP or ME for decarboxylated to form the isoprene unit, IPP. This unit is then growth. In the screening, we used ME and were able converted to DMAPP, with catalysis by IPP isomerase. HMG- to select ten mutants with the desired properties; the CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopen- tenyl diphosphate; DMAPP, dimethylallyl diphosphate. addition of ME, but not of DX, to the minimum medium facilitated the growth of the mutants.20) By complementation of these mutants, only the yaeM (DXP) by the condensation of pyruvate and D-glycer- gene, with an unknown function, was cloned.21) All aldehyde 3-phosphate, catalyzed by DXP synthase of the ten mutants that needed ME were conˆrmed to (Fig. 2).10) The dxs gene encoding this enzyme was be yaeM-gene-deˆcient mutants. ˆrst cloned from E. coli.11,12) This enzyme has a typi- Next, to check whether the yaeM gene was in fact cal thiamine-binding motif and needs both thiamine Mevalonate and Nonmevalonate Pathways 1621 Fig. 2. First Step of the Nonmevalonate Pathway. DXP synthase catalyzes the condensation of pyruvate and D-glyceraldehyde 3-phosphate to form DXP, the biosynthetic intermediate not only for IPP and DMAPP but also for thiamine and pydoxol. DX can be incorporated into E. coli and Arabidopsis thaliana to be used as the precursor of these compounds. TPP, thiamine diphosphate; DX, 1-deoxy-D-xylulose. Fig. 3. Two Reductoisomerase Reactions. (A) DXP reductoisomerase catalyzes simultaneous intramolecular rearrangement and reduction of DXP to form MEP, presumably via the hypothetical reaction intermediate, MEOP. FMM is a speciˆc inhibitor of this enzyme. ME can be incorporated into E. coli to be used as the precursor of IPP and DMAPP. (B) Ketol acid reductoisomerase in valine biosynthesis catalyzes the simultaneous in- tramolecular rearrangement and reduction of 2-acetolactate to form 2,3-dihydroxyisovalerate, presumably via a hypothetical reaction intermediate, 2-oxo-3-hydroxyisovalerate. This hypothetical compound inhibits ketol acid reductoisomerase activity. ME, 2-C- methylerythritol; MEOP, 2-C-methylerythrose 4-phosphate; FMM, fosmidomycin. responsible for MEP formation, we constructed a NADPH level, indicating that the enzyme converted plasmid for the overexpression of the gene product. DXP into an unknown reaction product. We puriˆed The puriˆed enzyme was detected as a homogeneous the reaction product and identiˆed it MEP on the protein band with a subunit size of 42 kDa by sodium basisofNMRandMSdata.20) The result showed that dodecyl sulfate-polyacrylamide gel electrophoresis. MEP is synthesized in the presence of NADPH by The enzyme had a molecular mass of 165 kDa as esti- rearrangement and reduction of DXP in a single step mated by native polyacrylamide gel electrophoresis. (Fig. 3(A)). We designated this enzyme DXP reduc- The enzyme appeared to be a homotetramer. toisomerase and renamed the yaeM gene ``dxr''.21) Incubation of the enzyme with DXP in the The reaction preceding this biosynthetic rearrange- presence of NADPH resulted in a decrease in the ment is involved in the biosynthesis of valine, isoleu- 1622 T.
Load more

Recommended publications
  • ASPP2 Inhibits Tumor Growth by Repressing the Mevalonate Pathway
    Liang et al. Cell Death and Disease (2019) 10:830 https://doi.org/10.1038/s41419-019-2054-7 Cell Death & Disease ARTICLE Open Access ASPP2 inhibits tumor growth by repressing the mevalonate pathway in hepatocellular carcinoma Beibei Liang1,RuiChen2, Shaohua Song3,HaoWang4,GuoweiSun5, Hao Yang1,WeiJing6, Xuyu Zhou6,ZhirenFu3, Gang Huang1 and Jian Zhao1 Abstract Cancer is, fundamentally, a disorder of cell growth and proliferation, which requires adequate supplies of energy and nutrients. In this study, we report that the haplo-insufficient tumor suppressor ASPP2, a p53 activator, negatively regulates the mevalonate pathway to mediate its inhibitory effect on tumor growth in hepatocellular carcinoma (HCC). Gene expression profile analysis revealed that the expression of key enzymes in the mevalonate pathway were increased when ASPP2 was downregulated. HCC cells gained higher cholesterol levels and enhanced tumor-initiating capability in response to the depletion of ASPP2. Simvastatin, a mevalonate pathway inhibitor, efficiently abrogated ASPP2 depletion-induced anchorage-independent cell proliferation, resistance to chemotherapy drugs in vitro, and tumor growth in xenografted nude mice. Mechanistically, ASPP2 interacts with SREBP-2 in the nucleus and restricts the transcriptional activity of SREBP-2 on its target genes, which include key enzymes involved in the mevalonate pathway. Moreover, clinical data revealed better prognosis in patients with high levels of ASPP2 and low levels of the mevalonate pathway enzyme HMGCR. Our findings provide functional and mechanistic insights into the critical role of ASPP2 in the regulation of the mevalonate pathway and the importance of this pathway in tumor initiation and tumor growth, which may provide a new therapeutic opportunity for HCC.
    [Show full text]
  • Enzyme Activity and Assays Introductory Article
    Enzyme Activity and Assays Introductory article Robert K Scopes, La Trobe University, Bundoora, Victoria, Australia Article Contents . Factors that Affect Enzymatic Analysis Enzyme activity refers to the general catalytic properties of an enzyme, and enzyme assays . Initial Rates and Steady State Turnover are standardized procedures for measuring the amounts of specific enzymes in a sample. Measurement of Enzyme Activity . Measurement of Protein Concentration Factors that Affect Enzymatic Analysis . Methods for Purifying Enzymes . Summary Enzyme activity is measured in vitro under conditions that often do not closely resemble those in vivo. The objective of measuring enzyme activity is normally to determine the exactly what the concentration is (some preparations of amount of enzyme present under defined conditions, so unusual substrates may be impure, or the exact amount that activity can be compared between one sample and present may not be known). This is because the rate another, and between one laboratory and another. The measured varies with substrate concentration more rapidly conditions chosen are usually at the optimum pH, as the substrate concentration decreases, as can be seen in ‘saturating’ substrate concentrations, and at a temperature Figure 1. In most cases an enzyme assay has already been that is convenient to control. In many cases the activity is established, and the substrate concentration, buffers and measured in the opposite direction to that of the enzyme’s other parameters used previously should be used again. natural function. Nevertheless, with a complete study of There are many enzymes which do not obey the simple the parameters that affect enzyme activity it should be Michaelis–Menten formula.
    [Show full text]
  • Opportunities for Catalysis in the 21St Century
    Opportunities for Catalysis in The 21st Century A Report from the Basic Energy Sciences Advisory Committee BASIC ENERGY SCIENCES ADVISORY COMMITTEE SUBPANEL WORKSHOP REPORT Opportunities for Catalysis in the 21st Century May 14-16, 2002 Workshop Chair Professor J. M. White University of Texas Writing Group Chair Professor John Bercaw California Institute of Technology This page is intentionally left blank. Contents Executive Summary........................................................................................... v A Grand Challenge....................................................................................................... v The Present Opportunity .............................................................................................. v The Importance of Catalysis Science to DOE.............................................................. vi A Recommendation for Increased Federal Investment in Catalysis Research............. vi I. Introduction................................................................................................ 1 A. Background, Structure, and Organization of the Workshop .................................. 1 B. Recent Advances in Experimental and Theoretical Methods ................................ 1 C. The Grand Challenge ............................................................................................. 2 D. Enabling Approaches for Progress in Catalysis ..................................................... 3 E. Consensus Observations and Recommendations..................................................
    [Show full text]
  • Against the Plasmodium Falciparum Apicoplast
    A Systematic In Silico Search for Target Similarity Identifies Several Approved Drugs with Potential Activity against the Plasmodium falciparum Apicoplast Nadlla Alves Bispo1, Richard Culleton2, Lourival Almeida Silva1, Pedro Cravo1,3* 1 Instituto de Patologia Tropical e Sau´de Pu´blica/Universidade Federal de Goia´s/Goiaˆnia, Brazil, 2 Malaria Unit/Institute of Tropical Medicine (NEKKEN)/Nagasaki University/ Nagasaki, Japan, 3 Centro de Mala´ria e Doenc¸as Tropicais.LA/IHMT/Universidade Nova de Lisboa/Lisboa, Portugal Abstract Most of the drugs in use against Plasmodium falciparum share similar modes of action and, consequently, there is a need to identify alternative potential drug targets. Here, we focus on the apicoplast, a malarial plastid-like organelle of algal source which evolved through secondary endosymbiosis. We undertake a systematic in silico target-based identification approach for detecting drugs already approved for clinical use in humans that may be able to interfere with the P. falciparum apicoplast. The P. falciparum genome database GeneDB was used to compile a list of <600 proteins containing apicoplast signal peptides. Each of these proteins was treated as a potential drug target and its predicted sequence was used to interrogate three different freely available databases (Therapeutic Target Database, DrugBank and STITCH3.1) that provide synoptic data on drugs and their primary or putative drug targets. We were able to identify several drugs that are expected to interact with forty-seven (47) peptides predicted to be involved in the biology of the P. falciparum apicoplast. Fifteen (15) of these putative targets are predicted to have affinity to drugs that are already approved for clinical use but have never been evaluated against malaria parasites.
    [Show full text]
  • Of Mevalonate Metabolism'
    ICANCER RESEARCH57. 3498—3505.AugustIS. 9971 Regulation of Proliferation and Ras Localization in Transformed Cells by Products of Mevalonate Metabolism' Jennifer A. Cuthbert2 and Peter E. Lipsky Department of Internal Medicine. The Unit'ersitv of Texas Southwestern Medical (‘enterat Dallas. Dallas. Texas 75235-9151 ABSTRACT position 186, the removal of the three COOH-terminal amino acids at positions 187—189, and carboxymethylation of the new COOH-termi Lovastatin, an inhibitor of 3-hydroxy.3-methylglutaryl (HMG) CoA nab cysteine. In addition, either palmitybation of other cysteine resi reductase, and 6-fluoromevalonate (Fmev), an inhibitor of diphospho dues in the COOH terminus (H-Ras, N-Ras, and K-RasA) or a mevalonate decarboxylase, blocked the synthesis of downstream meval. onate products, including prenyl-derived lipids, and prevented membrane pobybasic domain (K-RasB) is important in enhancing membrane localization of Ras in the myeloid cell line U.937. In contrast to lovastatin, association (7). These processes occur stepwise, and the first step, that which induced cytosol localization of Ras in U-937 cells, Fmev failed to of farnesybation of the full-length polypeptide, is thereby essential for increase cytosolic Ras and also completely prevented the proliferation of plasma membrane localization (12—14).Thus, compounds and muta U.937 cells. Growth of U-937 cells was restored by the addition of lovas tions that block the process of farnesylation interfere with the trans tatin to Fmev-blocked cells. These results implied that a product of formation and proliferation that are dependent upon mutationally mevalonate metabolism proximal to isopentenyl diphosphate was respon. activated Ras.
    [Show full text]
  • 33 34 35 Lipid Synthesis Laptop
    BI/CH 422/622 Liver cytosol ANABOLISM OUTLINE: Photosynthesis Carbohydrate Biosynthesis in Animals Biosynthesis of Fatty Acids and Lipids Fatty Acids Triacylglycerides contrasts Membrane lipids location & transport Glycerophospholipids Synthesis Sphingolipids acetyl-CoA carboxylase Isoprene lipids: fatty acid synthase Ketone Bodies ACP priming 4 steps Cholesterol Control of fatty acid metabolism isoprene synth. ACC Joining Reciprocal control of b-ox Cholesterol Synth. Diversification of fatty acids Fates Eicosanoids Cholesterol esters Bile acids Prostaglandins,Thromboxanes, Steroid Hormones and Leukotrienes Metabolism & transport Control ANABOLISM II: Biosynthesis of Fatty Acids & Lipids Lipid Fat Biosynthesis Catabolism Fatty Acid Fatty Acid Synthesis Degradation Ketone body Utilization Isoprene Biosynthesis 1 Cholesterol and Steroid Biosynthesis mevalonate kinase Mevalonate to Activated Isoprenes • Two phosphates are transferred stepwise from ATP to mevalonate. • A third phosphate from ATP is added at the hydroxyl, followed by decarboxylation and elimination catalyzed by pyrophospho- mevalonate decarboxylase creates a pyrophosphorylated 5-C product: D3-isopentyl pyrophosphate (IPP) (isoprene). • Isomerization to a second isoprene dimethylallylpyrophosphate (DMAPP) gives two activated isoprene IPP compounds that act as precursors for D3-isopentyl pyrophosphate Isopentyl-D-pyrophosphate all of the other lipids in this class isomerase DMAPP Cholesterol and Steroid Biosynthesis mevalonate kinase Mevalonate to Activated Isoprenes • Two phosphates
    [Show full text]
  • Recitation Section 4 Answer Key Biochemistry—Energy and Glycolysis
    MIT Department of Biology 7.014 Introductory Biology, Spring 2005 Recitation Section 4 Answer Key February 14-15, 2005 Biochemistry—Energy and Glycolysis A. Why do we care In lecture we discussed the three properties of a living organism: metabolism, regulated growth, and replication. Today we will focus on metabolism and biosynthesis. 1. It was said in lecture that chemical reactions are the basis of life. Why do we say that? Being alive implies being able to change your state in response to a change in internal or environmental conditions. We discussed previously that any change in the observable characteristics of a cell begins as and is propagated by molecular interactions. Molecular interactions propagate the signal by changing the state of molecules, i.e. by reactions. 2. Why is metabolism required for life? A cell is subject to all laws of chemistry and physics, including the first and second law of thermodynamics. Changing the state of molecules dissipates energy along the way, so getting or making new energy is essential to life. The energy is then used for many purposes, including making the building blocks and precursors and then using them to build macromolecules that make up cells—biosynthesis. 3. Can an entity that performs no chemical reactions be considered “alive?” In general, no. But there are special cases of the cells that do not perform reactions right at the moment, but have the potential to perform reactions if they encounter particular conditions. Some examples of such cells are spores in nature or frozen permanents in laboratory. If they experience certain conditions, such as availability of food for spores, or defrosting and food source for frozen permanents, these cells will again perform metabolism.
    [Show full text]
  • 2.4 the Breakdown Chemical Reactions Bonds Break and Form During Chemical Reactions
    DO NOT EDIT--Changes must be made through “File info” CorrectionKey=B 2.4 Chemical Reactions KEY CONCEPT VOCABULARY Life depends on chemical reactions. chemical reaction MAIN IDEAS reactant Bonds break and form during chemical reactions. product Chemical reactions release or absorb energy. bond energy equilibrium activation energy Connect to Your World exothermic When you hear the term chemical reaction, what comes to mind? Maybe you think endothermic of liquids bubbling in beakers. You probably do not think of the air in your breath, but most of the carbon dioxide and water vapor that you breathe out are made by chemical reactions in your cells. MAIN IDEA Bonds break and form during chemical reactions. Plant cells make cellulose by linking simple sugars together. Plant and animal cells break down sugars to get usable energy. And all cells build protein mol- ecules by bonding amino acids together. These are just a few of the chemical reactions in living things. Chemical reactions change substances into different substances by breaking and forming chemical bonds. Although the matter changes form, both matter and energy are conserved in a chemical reaction. Reactants, Products, and Bond Energy Your cells need the oxygen molecules that you breathe in. Oxygen (O2) plays a part in a series of chemical reactions that provides usable energy for your cells. These reactions, which are described in detail in the chapter Cells and Energy, break down the simple sugar glucose (C6H12O6). The process uses oxygen and glucose and results in carbon dioxide (CO2), water (H2O), and usable energy. Oxygen and glucose are the reactants.
    [Show full text]
  • The NTP Generating Activity of Pyruvate Kinase II Is Critical
    RESEARCH ARTICLE The NTP generating activity of pyruvate kinase II is critical for apicoplast maintenance in Plasmodium falciparum Russell P Swift, Krithika Rajaram, Cyrianne Keutcha, Hans B Liu, Bobby Kwan, Amanda Dziedzic, Anne E Jedlicka, Sean T Prigge* Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, United States Abstract The apicoplast of Plasmodium falciparum parasites is believed to rely on the import of three-carbon phosphate compounds for use in organelle anabolic pathways, in addition to the generation of energy and reducing power within the organelle. We generated a series of genetic deletions in an apicoplast metabolic bypass line to determine which genes involved in apicoplast carbon metabolism are required for blood-stage parasite survival and organelle maintenance. We found that pyruvate kinase II (PyrKII) is essential for organelle maintenance, but that production of pyruvate by PyrKII is not responsible for this phenomenon. Enzymatic characterization of PyrKII revealed activity against all NDPs and dNDPs tested, suggesting that it may be capable of generating a broad range of nucleotide triphosphates. Conditional mislocalization of PyrKII resulted in decreased transcript levels within the apicoplast that preceded organelle disruption, suggesting that PyrKII is required for organelle maintenance due to its role in nucleotide triphosphate generation. Introduction *For correspondence: With increasing resistance to current front-line antimalarials, there is a crucial need to find new thera- [email protected] peutic interventions with novel mechanisms of action (Dondorp et al., 2009; Trape, 2001). The api- coplast organelle within the parasite has often been considered as a source of new drug targets Competing interests: The since it is required for blood-stage survival, in addition to possessing evolutionarily distinct biochem- authors declare that no ical pathways that are not present in the human host (Goodman and McFadden, 2013; competing interests exist.
    [Show full text]
  • Construction and Optimization of Mevalonate Pathway for Production Of
    Construction and Optimization of Mevalonate Pathway for Production of Isoprenoids in Escherichia coli by Farnaz Nowroozi A dissertation submitted in partial satisfaction of the requirements for the degree of Joint Doctor of Philosophy with University of California, San Francisco in Engineering-Bioengineering in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Jay D. Keasling, Chair Professor Adam P. Arkin Professor Francis C. Szoka Professor Marc K. Hellerstein Fall 2009 1 The dissertation of Farnaz Foroughi-Boroujeni Nowroozi, titled Construction and Optimization of Mevalonate Pathway for production of Isoprenoids in Escherichia coli , is approved: Chair _______________________________ Date ____________________ _______________________________ Date____________________ _______________________________ Date ____________________ _______________________________ Date ____________________ University of California, Berkeley 2 Abstract Construction and Optimization of Mevalonate Pathway for production of Isoprenoids in Escherichia coli by Farnaz Foroughi-Boroujeni Nowroozi Doctor of Philosophy in Bioengineering University of California, Berkeley Professor Jay D. Keasling, Chair The isoprenoid family, containing over 50,000 members, constitutes one of the most structurally diverse groups of natural products. They range from essential and relatively universal primary metabolites, such as sterols, carotenoids, and hormones, to more unique secondary metabolites that serve roles in plant defense and communication and cellular and organismal development. Although these molecules have vast potential in medicine and industry their production is limited by two factors: 1- The yields from harvest and extraction of these compounds from their native sources are low 2- Due to their complex structure, synthetic routes to most isoprenoids are difficult and inefficient Therefore engineering metabolic pathways for production of large quantities of isoprenoids in a microbial host is an attractive approach.
    [Show full text]
  • Engineering a Mevalonate Pathway in Escherichia Coli for Production of Terpenoids
    ARTICLES Engineering a mevalonate pathway in Escherichia coli for production of terpenoids Vincent JJ Martin1,2,3, Douglas J Pitera1,3,Sydnor T Withers1,Jack D Newman1 & Jay D Keasling1 Isoprenoids are the most numerous and structurally diverse family of natural products. Terpenoids, a class of isoprenoids often isolated from plants, are used as commercial flavor and fragrance compounds and antimalarial or anticancer drugs. Because plant tissue extractions typically yield low terpenoid concentrations, we sought an alternative method to produce high-value terpenoid compounds, such as the antimalarial drug artemisinin, in a microbial host. We engineered the expression of a synthetic amorpha-4,11-diene synthase gene and the mevalonate isoprenoid pathway from Saccharomyces cerevisiae in Escherichia coli. Concentrations of amorphadiene, the sesquiterpene olefin precursor to artemisinin, reached 24 µg caryophyllene equivalent/ml. Because isopentenyl and dimethylallyl pyrophosphates are the universal precursors to all isoprenoids, the strains developed in this study can serve as platform hosts for the production of any terpenoid compound for which a terpene synthase gene is available. http://www.nature.com/naturebiotechnology Terpenoids comprise a highly diverse class of natural products from certain cancers10,11, and irufloven, a third-generation semisynthetic which numerous commercial flavors, fragrances and medicines are analog of the sesquiterpene illudin S that are in late-stage clinical derived. These valuable compounds are commonly isolated from trials for the treatment of various refractory and relapsed can- plants, microbes and marine organisms. For example, terpenoids cers12,13.In general, these drugs are extracted from the host plant, in extracted from plants are used as anticancer and antimalarial which they accumulate in very small amounts, before further drugs1,2.Because these compounds are naturally produced in small derivatization or use.
    [Show full text]
  • Enzyme Kinetics in This Exercise We Will Look at the Catalytic Behavior of Enzymes
    1 Enzyme Kinetics In this exercise we will look at the catalytic behavior of enzymes. You will use Excel to answer the questions in the exercise section. At the end of this session, you must hand in answers to all the questions, along with print outs of any plots you created. Background Enzymes are the catalysts of biological systems and are extremely efficient and specific as catalysts. In fact, typically, an enzyme accelerates the rate of a reaction by factors of at least a million compared to the rate of the same reaction in the absence of the enzyme. Most biological reactions do not occur at perceptible rates in the absence of enzymes. One of the simplest biological reactions catalyzed by an enzyme is the hydration of CO2. The catalyst in this reaction is carbonic anhydrase. This reaction is part of the respiration cycle which expels CO2 from the body. Carbonic anhydrase is a highly efficient enzyme – each enzyme molecule 5 can catalyze the hydration of 10 CO2 molecules per second. Enzymes are highly specific. Typically a particular enzyme catalyzes only a single chemical reaction or a set of closely related chemical reactions. As is true of any catalyst, enzymes do not alter the equilibrium point of the reaction. This means that the enzyme accelerates the forward and reverse reaction by precisely the same factor. For example, consider the interconversion of A and B. A ↔ B (1) -4 -1 Suppose that in the absence of the enzyme the forward rate constant (kf) is 10 s and the -6 -1 reverse rate constant (kr) is 10 s .
    [Show full text]