Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors

The essential steps of the novel non-mevalonate pathway of isopentenyl diphosphate and isoprenoid biosynthesis in plants are described. The first five enzymes and genes of this 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway are known. The herbicide fosmidomycin specifically blocks the second enzyme, the DOXP reductoisomerase. The DOXP/MEP pathway is also present in several pathogenic bacteria and the malaria parasite. Hence, all herbicides and inhibitors blocking this novel isoprenoid pathway in plants are also potential drugs against malaria and diseases caused by pathogenic bacteria.     

Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway

Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.     

Vγ9/Vδ2 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.     

辅酶Q10生物合成与生产研究进展

微生物发酵法是生产辅酶Q10的最佳工艺.辅酶Q10的生物合成途径包括异戊二烯焦磷酸合成、聚十异戊二烯焦磷酸合成、苯环修饰等过程.1-脱氧-D-木酮糖-5-磷酸合成酶、聚十异戊二烯焦磷酸合成酶、对羟基笨甲酸聚十异戊二烯焦磷酸转移酶等是Q10合成的关键酶.生产辅酶Q10的菌种可通过诱变、基因重组和支路敲除等方法获得.氧化还原电位控制、pH控制补料分批发酵、发酵萃取耦合技术等新工艺逐浙应用于辅酶Q10生产.     

1-Deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-xylulose 5-phosphate synthase A and B, containing a His(6) tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-xylulose 5-phosphate synthase A and B: K(m)(pyruvate) = 0.61 and 3.0 mM, K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 microM, and V(max) = 1.9 and 1.4 micromol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-D-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.     

Structure and mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. An enzyme in the mevalonate-independent isoprenoid biosynthetic pathway.

The enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to MECDP, a highly unusual cyclodiphosphate-containing intermediate on the mevalonate-independent pathway to isopentenyl diphosphate and dimethylallyl diphosphate. We now report two x-ray crystal structures of MECDP synthase refined to 2.8-A resolution. The first structure contains a bound Mn(2+) cation, and the second structure contains CMP, MECDP, and Mn(2+). The protein adopts a homotrimeric quaternary structure built around a central hydrophobic cavity and three externally facing active sites. Each of these active sites is located between two adjacent monomers. A tetrahedrally arranged transition metal binding site, potentially occupied by Mn(2+), sits at the base of the active site cleft. A phosphate oxygen of MECDP and the side chains of Asp(8), His(10), and His(42) occupy the metal ion coordination sphere. These structures reveal for the first time the structural determinants underlying substrate, product, and Mn(2+) recognition and the likely catalytic mechanism accompanying the biosynthesis of the cyclodiphosphate-containing isoprenoid precursor, MECDP.     

LytB, a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli.

The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.     

Cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural-rubber producing plant, Hevea brasiliensis

Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.     

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.     

Identification of gcpE as a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis in Escherichia coli

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most eubacteria and plants and has remarkable biotechnological interest. However, only the first steps of this pathway have been determined. Using bioinformatic and genetic approaches, we have identified gcpE as a novel gene of the MEP pathway. The distribution of this gene in bacteria and plants strictly parallels that of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, which catalyses the first committed step of the MEP pathway. Our data demonstrate that the gcpE gene is essential for the MEP pathway in Escherichia coli and indicate that this gene is required for the trunk line of the isoprenoid biosynthetic route.     

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.     

Expression of the farnesyldiphosphate synthase gene of Saccharomyces cerevisiae in tobacco

The coding region of the farnesyldiphosphate synthase (FDP synthase) gene from Saccharomyces cerevisiae has been inserted into a pBin19 vector, downstream of the cauliflower mosaic virus (CaMV) 35S promoter, in order to allow its expression in the genome of a higher plant, Nicotiana tabacum. We have produced transgenic tobacco in which the expression of the foreign gene leads to functional FDP synthase activity. In these transgenic plants, total FDP synthase-specific activity is increased 12-fold compared to controls. This increase of FDP synthase activity has been correlated to a clear increase of both sterol and carotenoid biosynthesis. This heterologous expression is also related to an increased resistance of transformed plants to R172117, a specific inhibitor of FDP synthase, and to sterol biosynthesis inhibitors such as flusilazol and fenpropimorph.Abbreviations: AP, Annick Petit; BAP, benzylaminopurine; CaMV, cauliflower mosaic virus; CTAB, cetyltrimethylammonium bromide; DMAEDP, dimethylamino ethyl diphosphate; DMADP, dimethylallyl diphosphate; DTT, dithiothreitol; ERG12, mevalonate kinase yeast gene; ERG20, FDP synthase yeast gene; FDP, farnesyl diphosphate; GGDP, geranylgeranyl diphosphate; GDP, geranyl diphosphate; IDP, isopentenyl diphosphate; LB, Luria Bertani; MS, Murashige and Skoog; NAA, Naphtaleneacetic acid; PVP, polyvinyl pyrrolidone; SBI, sterol biosynthesis inhibitor.     

Transport of isoprenoid intermediates across chloroplast envelope membranes

The common precursor for isoprenoid biosynthesis in plants, isopentenyl diphosphate (IPP), is synthesized by two pathways, the cytosolic mevalonate pathway and the plastidic 1-deoxy-D-xylulose 5-phosphate/methylerythritol phosphate (DOXP/MEP) pathway. The DOXP/MEP pathway leads to the formation of various phosphorylated intermediates, including DOXP, 4-hydroxy-3-methylbutenyl diphosphate (HMBPP), and finally IPP. There is ample evidence for metabolic cross-talk between the two biosynthetic pathways. The present study addresses the question whether isoprenoid intermediates could be exchanged between both compartments by members of the plastidic phosphate translocator (PT) family that all mediate a counter-exchange between inorganic phosphate and various phosphorylated compounds. Transport experiments using intact chloroplasts, liposomes containing reconstituted envelope membrane proteins or recombinant PT proteins showed that HMBPP is not exchanged between the cytosol and the chloroplasts and that the transport of DOXP is preferentially mediated by the recently discovered plastidic transporter for pentose phosphates, the xylulose 5-phosphate translocator. Evidence is presented that transport of IPP does not proceed via the plastidic PTs although IPP transport is strictly dependent on various phosphorylated compounds on the opposite side of the membrane. These phosphorylated trans compounds are, in part, also used as counter-substrates by the plastidic PTs but appear to only trans activate IPP transport without being transported.     

Biosynthesis of 2-methyl-3-buten-2-ol emitted from needles of Pinus ponderosa via the non-mevalonate DOXP/MEP pathway of isoprenoid formation

The volatile hemiterpene 2-methyl-3-buten-2-ol (MBO) is emitted from the needles of several pine species from the Western United States and contributes to ozone formation in the atmosphere. It is synthesised enzymatically from dimethylallyl diphosphate (DMAPP). We show here that needles of Pinus ponderosa Laws. incorporated [1-2H1]-1-deoxy-D-xylulose (d-DOX) into the emitted MBO, but not D,L-[2-13C]mevalonic acid lactone. Furthermore, MBO emission was inhibited by fosmidomycin, a specific inhibitor of the second enzyme of the mevalonate-independent pathway of isopentenyl diphosphate and DMAPP formation, i.e. the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. We thus prove that MBO emitted from needles of P. ponderosa is primarily formed via the DOXP/MEP pathway.     

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).     

类异戊二烯非甲羟戊酸代谢途径的分子生物学研究进展

近期发现的类异戊二烯非甲羟戊酸代谢途径是类异戊二烯化合物生成合成的另一途径。文章对该代谢途径的分子生物学研究进展进行了综述。重点介绍非甲羟戊酸代谢途径的发现和5－磷酸脱氧木糖合成酶、5－磷酸脱氧木糖还原异构醇、异戊二烯焦磷酸合成酶的分子克隆的最新进展以及非甲羟戊酸代谢途径的发现在农业和医药等领域的应用。     

Characterization of the Mycobacterium tuberculosis 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase: potential for drug development

Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-D-erythritol is formed from 2-C-methyl-D-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5'-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 microM for MEP and 53.2 microM for CTP. Calculated kcat and kcat/Km values were 0.72 min(-1) and 12.3 mM(-1) min(-1) for MEP and 1.0 min(-1) and 18.8 mM(-1) min(-1) for CTP, respectively.