青蒿素生物合成分子调控研究进展

青蒿素是目前世界上最有效的疟疾治疗药物。通过对青蒿素的生物合成途径,青蒿素生物合成途径的关键酶,青蒿素生物合成的分子调控的介绍,综述了青蒿素生物合成分子调控的最新研究进展。     

Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-delta-cadinene synthase

Cotton plants were transformed with an antisense construct of cdn1-Cl, a member of a complex gene family of delta-(+)cadinene (CDN) synthase. This synthase catalyzes the cyclization of (E,E)-farnesyl diphosphate to form CDN, and in cotton, it occupies the committed step in the biosynthesis of cadinane sesquiterpenoids and heliocides (sesterterpenoids). Southern analyses of the digestion of leaf DNA from R(o), T(o), and T(1) plants with Hind III, Pst I and Kpn I restriction enzymes show the integration of antisense cdn1-C1 cDNA driven by the CaMV 35S promoter into the cotton genome. Northern blots demonstrate the appearance of cdn synthase mRNA preceding CDN synthase activity and the formation of gossypol in developing cottonseed. T(2) cottonseed show a reduced CDN synthase activity and up to a 70% reduction in gossypol. In T(1) leaves the accumulated amounts of gossypol, hemigossypolone and heliocides are reduced 92.4, 83.3 and 68.4%, respectively. These data demonstrate that the integration of antisense cdn1-C1 cDNA into the cotton genome leads to a reduction of CDN synthase activity and negatively impacts on the biosynthesis of cadinane sesquiterpenoids and heliocides in cotton plants.     

高等植物脱落酸生物合成途径及其酶调控

脱落酸（ABA）生物合成一般有两条途径：C15直接途径和C40间接途径, 前者经C15法呢焦磷酸（FPP）直接形成ABA;后者经由类胡萝卜素的氧化裂解间接形成ABA, 是高等植物ABA生物合成的主要途径。9-顺式环氧类胡萝卜素氧化裂解为黄质醛是植物ABA生物合成的关键步骤, 然后黄质醛被氧化形成一种酮, 该过程需NAD为辅因子, 酮再转变形成ABA-醛, ABA-醛氧化最终形成ABA。在该途径中,玉米黄质环氧化酶（ZEP）、9-顺式环氧类胡萝卜素双加氧酶（NCED）和醛氧化酶（AO）可能起重要作用。     

法呢基焦磷酸(FPP)的生物合成及其分子调控

法呢基焦磷酸合酶作为异戊二烯途径中的重要调节酶,是许多萜类物质的合成前体。FPS的cDNA克隆在许多生物体中也已得到了分离并进行了表达特性研究。从FPP的生物合成途径入手,对FPP生物学特性、FPS酶基因调控的相关信息进行了综述,同时对FPS在基因工程方面的应用进行了展望。     

Farnesyl diphosphate synthase is a cytosolic enzyme in Leishmania major promastigotes and its overexpression confers resistance to risedronate

Farnesyl diphosphate synthase is the most likely molecular target of aminobisphosphonates (e.g., risedronate), a set of compounds that have been shown to have antiprotozoal activity both in vitro and in vivo. This protein, together with other enzymes involved in isoprenoid biosynthesis, is an attractive drug target, yet little is known about the compartmentalization of the biosynthetic pathway. Here we show the intracellular localization of the enzyme in wild-type Leishmania major promastigote cells and in transfectants overexpressing farnesyl diphosphate synthase by using purified antibodies generated towards a homogenous recombinant Leishmania major farnesyl diphosphate synthase protein. Indirect immunofluorescence, together with immunoelectron microscopy, indicated that the enzyme is mainly located in the cytoplasm of both wild-type cells and transfectants. Digitonin titration experiments also confirmed this observation. Hence, while the initial step of isoprenoid biosynthesis catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase is located in the mitochondrion, synthesis of farnesyl diphosphate by farnesyl diphosphate synthase is a cytosolic process. Leishmania major promastigote transfectants overexpressing farnesyl diphosphate synthase were highly resistant to risedronate, and the degree of resistance correlated with the increase in enzyme activity. Likewise, when resistance was induced by stepwise selection with the drug, the resulting resistant promastigotes exhibited increased levels of farnesyl diphosphate synthase. The overproduction of protein under different conditions of exposure to risedronate further supports the hypothesis that this enzyme is the main target of aminobisphosphonates in Leishmania cells.     

Farnesyl pyrophosphate synthase: a key enzyme in isoprenoid biosynthetic pathway and potential molecular target for drug development

As isoprenoid biosynthetic pathway has gained importance since last few years, key enzymes of this pathway have been characterized and their functional roles in the cell metabolism have been explored using molecular biology approaches. A key enzyme in this pathway is farnesyl pyrophosphate (EC 2.5.1.10) synthase (FPPS) which supplies precursors for the biosynthesis of essential isoprenoids like carotenoids, withanolides, ubiquinones, dolichols, sterols, among others and also helps in farnesylation and geranylation of proteins. It is a chain elongation enzyme which catalyzes head to tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl pyrophosphate (FPP). Recent studies have validated FPPS as a molecular target of bisphosphonates for drug development against tumors as well as human pathogens. The present paper synthesizes the information on characterization, structural and functional relationships, evolution, localization as well as advances on FPPS enzyme as a target for drug development.     

Identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes. AA-CoA thiolase, hmg-coa synthase, MPPD, and FPP synthase

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. The peroxisomal targeting signals for phosphomevalonate kinase and isopentenyl diphosphate isomerase have been identified. In the current study we identify the peroxisomal targeting signals required for four other enzymes of the cholesterol biosynthetic pathway: acetoacetyl-CoA (AA-CoA) thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, mevalonate diphosphate decarboxylase (MPPD), and farnesyl diphosphate (FPP) synthase. Data are presented that demonstrate that mitochondrial AA-CoA thiolase contains both a mitochondrial targeting signal at the amino terminus and a peroxisomal targeting signal (PTS-1) at the carboxy terminus. We also analyze a new variation of PTS-2 sequences required to target HMG-CoA synthase and MPPD to peroxisomes. In addition, we show that FPP synthase import into peroxisomes is dependent on the PTS-2 receptor and identify at the amino terminus of the protein a 20-amino acid region that is required for the peroxisomal localization of the enzyme.These data provide further support for the conclusion that peroxisomes play a critical role in cholesterol biosynthesis.     

Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.     

Subcellular evidence for the involvement of peroxisomes in plant isoprenoid biosynthesis

The role of peroxisomes in isoprenoid metabolism, especially in plants, has been questioned in several reports. A recent study of Sapir-Mir et al.¹ revealed that the two isoforms of isopentenyl diphosphate (IPP) isomerase, catalyzing the isomerisation of IPP to dimethylallyl diphosphate (DMAPP) are found in the peroxisome. In this addendum, we provide additional data describing the peroxisomal localization of 5-phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase, the last two enzymes of the mevalonic acid pathway leading to IPP.² This finding was reinforced in our latest report showing that a short isoform of farnesyl diphosphate, using IPP and DMAPP as substrates, is also targeted to the organelle.³ Therefore, the classical sequestration of isoprenoid biosynthesis between plastids and cytosol/ER can be revisited by including the peroxisome as an additional isoprenoid biosynthetic compartment within plant cells.     

棉酚及其衍生物合成途径的研究进展

棉酚作为一种倍半萜烯类植物抗菌素广泛存在于棉花的根、种子等表皮组织的色素腺体里。该化合物已在医药、工业和农业等领域得到广泛应用。近来对棉酚及其衍生物合成途径的研究日趋深入,许多关键酶基因已得到克隆和分析。掌握棉酚及其衍生物的生物合成途径,并试图通过基因工程改造的方法来控制植物体棉酚的合成具有重要意义。概述了棉酚及其衍生物合成途径和关键酶的研究进展并展望其应用前景。     

Biosynthesis of carotenoids in plastids of plants

This review deals with various aspects of the biosynthesis of carotenoids in chromoplasts and chloroplasts of green algae and higher plants. Two pathways of biosynthesis of the key C5-isoprene units are considered: 1) from acetate via mevalonate (C6) followed by its enzymatic conversions to isopentenyl diphosphate (C5); 2) from glucose via formation of glyceraldehyde-3-phosphate (C3) and pyruvate and their condensation via intermediary products to isopentenyl diphosphate (C5). Subsequent biosynthesis of carotenoids from isopentenyl diphosphate (C5) and dimethylallyl diphosphate (C5) involves a common route including their conversion into geranyl diphosphate (C10), farnesyl diphosphate (C15), geranylgeranyl diphosphate (C20), and synthesis of phytoene (C40). All stages of phytoene desaturation accompanied by formation of acyclic compounds such as zeta-carotene, neurosporene, and lycopene and their cyclization to alpha-, beta-, and epsilon-carotenes are considered in detail. Formation of xanthophylls in chloroplasts and chromoplasts involves sequential oxidations yielding hydroxy, epoxy, and oxo groups. Genetic control of biosynthesis of carotenoids is considered.     

Thiamine in plants: Aspects of its metabolism and functions

Thiamine diphosphate (vitamin B₁) plays a fundamental role as an enzymatic cofactor in universal metabolic pathways including glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle. In addition, thiamine diphosphate has recently been shown to have functions other than as a cofactor in response to abiotic and biotic stress in plants. Recently, several steps of the plant thiamine biosynthetic pathway have been characterized, and a mechanism of feedback regulation of thiamine biosynthesis via riboswitch has been unraveled. This review focuses on these most recent advances made in our understanding of thiamine metabolism and functions in plants. Phenotypes of plant mutants affected in thiamine biosynthesis are described, and genomics, proteomics, and metabolomics data that have increased further our knowledge of plant thiamine metabolic pathways and functions are summarized. Aspects of thiamine metabolism such as catabolism, salvage, and transport in plants are discussed.     

Current state of carotenoid biosynthesis in chloroplasts of eucaryotes

The author discusses the current state of biochemical and genetic aspects of carotenoid biosynthesis in chloroplasts of algae and higher plants. Two ways of biosynthesis of key C5-isoperene units have been considered: 1) from acetate (C2) via mevalonic acid (C6) and its enzymatic conversions up to isopenthenyl diphosphate (C5) and 2) from glucose (C6) to formation of glyceraldehyde-3-phosphate (C3), to piruvate and their condensation via intermediate products up to isopenthenyl diphosphate (C5). Further biosynthesis of carotenoids from isopenthenyl diphosphate (C5) and dimethylallyl diphosphate (C5) in every organism is effected by the common scheme with further conservation of them up to geranyl diphosphate (C10), farnesyl diphosphate (C15), geranylgeranyl diphosphate (C20) and synthesis of phytoene (C40). All stages of phytoene desaturation up to formation of acyclic compounds are discussed. It is shown how in the process of subsequent oxidation and formation of hydroxy-, epoxy- and oxo-groups cyclic xanthophylls in chloroplasts of plants and algae are formed. Genetic control over biosynthesis of carotenoids is discussed.