Leishmania amazonensis: biosynthesis of polyprenols of 9 isoprene units by amastigotes

The isoprenoid metabolic pathway in protozoa of the Leishmania genus exhibits distinctive characteristics. These parasites, as well as other members of the Trypanosomatidae family, synthesize ergosterol, instead of cholesterol, as the main membrane sterol lipid. Leishmania has been shown to utilize leucine, instead of acetate as the main precursor for sterol biosynthesis. While mammalian dolichols are molecules containing 15-23 isoprene units, Leishmania amazonensis promastigotes synthesize dolichol of 11 and 12 units. In this paper, we show that the intracellular stages of L. amazonensis, amastigotes, synthesize mainly polyprenols of 9 isoprene units, instead of dolichol.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H⁺ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H⁺-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H⁺ in the course of cellular amino acid uptake by PAT1.     

Biosynthesis of mono- and sesquiterpenes in peppermint from mevalonate-2-C

The volatile oil from peppermint (Mentha piperita L.) is composed primarily of monoterpenes with less than 2% sesquiterpenes. However, radioactivity from mevalonate-2-¹⁴C is incorporated into caryophyllene and other sesquiterpene hydrocarbons much more extensively than into monoterpenes by peppermint cuttings. Both mono- and sesquiterpenes show maximum incorporation of label after 6 hr (0·03% vs. 0·33% of the physiological isomer) and lose 75% of the incorporated label after an additional 6 hr. Caryophyllene derived from mevalonate-2-¹⁴C after 6 hr of incorporation was chemically degraded. The isoprenoid origin of caryophyllene was confirmed, and preferential labelling of the isopentenyl pyrophosphate derived portions of the molecule was noted. On the basis of such evidence it appears that separate sites may exist for the biosynthesis of mono- and sesquiterpenes and that an endogenous dimethylallyl pyrophosphate pool may participate in the biosynthesis of sesquiterpenes in peppermint.     

Stereospecificity of biosynthesis of triterpene alcohols in Calendula officinalis flowers

Flowers of Calendula officinalis were incubated with mevalonic acid doubly labelled with ¹⁴C in position 2 and ³H in positions 2R, 2S, 4R or 5R,S and the [³H/¹⁴C] ratios determined in squalene and pentacylic mono- and dihydroxy-triterpene alcohols and also in some derivatives prepared from the triterpene alcohols. ³H atoms were located in positions 3, 12, 16, 21, 29, 30 of the ursane skeleton, positions 3, 12, 29, 30 of the lupane skeleton and positions 3, 11, 12, 18 of the oleanane skeleton. Stabilization of α- and β-Amyrins, ω-taraxasterol and lupeol occurs with the elimination of a proton from positions 12, 21 and 29 (or 30) respectively. In addition, during hydroxylation of triterpene monols to the corresponding diols a proton is substituted by the hydroxyl group.     

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。     

Characterization of the complete chloroplast genome of Hevea brasiliensis reveals genome rearrangement, RNA editing sites and phylogenetic relationships

Rubber tree (Hevea brasiliensis) is an economical plant and widely grown for natural rubber production. However, genomic research of rubber tree has lagged behind other species in the Euphorbiaceae family. We report the complete chloroplast genome sequence of rubber tree as being 161,191 bp in length including a pair of inverted repeats of 26,810 bp separated by a small single copy region of 18,362 bp and a large single copy region of 89,209 bp. The chloroplast genome contains 112 unique genes, 16 of which are duplicated in the inverted repeat. Of the 112 unique genes, 78 are predicted protein-coding genes, 4 are ribosomal RNA genes and 30 are tRNA genes. Relative to other plant chloroplast genomes, we observed a unique rearrangement in the rubber tree chloroplast genome: a 30-kb inversion between the trnE(UUC)-trnS(GCU) and the trnT(GGU)-trnR(UCU). A comparison between the rubber tree chloroplast genes and cDNA sequences revealed 51 RNA editing sites in which most (48 sites) were located in 26 protein coding genes and the other 3 sites were in introns. Phylogenetic analysis based on chloroplast genes demonstrated a close relationship between Hevea and Manihot in Euphorbiaceae and provided a strong support for a monophyletic group of the eurosid I.     

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.     

Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959 bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67 kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza.