A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni)

Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA₃) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.     

Plant isoprenoid biosynthesis via the MEP pathway: In vivo IPP/DMAPP ratio produced by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase in tobacco BY-2 cell cultures

Feeding tobacco BY-2 cells with [2-¹³C,4-²H]deoxyxylulose revealed from the ¹³C labeling that the plastid isoprenoids, synthesized via the MEP pathway, are essentially derived from the labeled precursor. The ca. 15% ²H retention observed in all isoprene units corresponds to the isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) ratio (85:15) directly produced by the hydroxymethylbutenyl diphosphate reductase, the last enzyme of the MEP pathway. ²H retention characterizes the isoprene units derived from the DMAPP branch, whereas ²H loss represents the signature of the IPP branch. Taking into account the enantioselectivity of the reactions catalyzed by the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase, the IPP isomerase and the trans-prenyl transferase, a single biogenetic scheme allows to interpret all labeling patterns observed in bacteria or plants upon incubation with ²H labeled deoxyxylulose.     

Chromones from the tubers of Eranthis cilicica and their antioxidant activity

Chemical studies on the constituents of Eranthis cilicica led to isolation of ten chromone derivatives, two of which were previously known. Comprehensive spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of enzymatic hydrolysis allowed the chemical structures of the compounds to be assigned as 8,11-dihydro-5-hydroxy-2,9-dihydroxymethyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 5,7-dihydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 5,7-dihydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 7-[(β-d-glucopyranosyl)oxy]-5-hydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 7-[(β-d-glucopyranosyl)oxy]-5-hydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 9-[(O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)oxy]methyl-8,11-dihydro-5,9-dihydroxy-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 8,11-dihydro-5,9-dihydroxy-9-hydroxymethyl-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, and 7-[(O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)oxy]methyl-4-hydroxy-5H-furo[3,2-g][1]benzopyran-5-one, respectively. The isolated compounds were evaluated for their antioxidant activity.     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.     

Structure of the (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase from Plasmodium falciparum

Terpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe–2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs.     

Cloning and expression analysis of ten genes associated with picrosides biosynthesis in Picrorhiza kurrooa

Picrorhiza kurrooa Royle ex Benth. is an economically important medicinal plant known to yield picrosides which have high medicinal value. Picroside I and picroside II are major picrosides associated with various bioactivities. The present work analyzed the expression of various genes of the picrosides biosynthesis pathway in different tissues of the plant in relation to the picrosides content. Eight full-length cDNA sequences namely, 1-deoxy-d-xylulose-5-phosphate synthase (2.317 kb), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (1.767 kb), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (1.674 kb), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (1.701 kb), acetyl-CoA acetyltransferase (1.545 kb), 3-hydroxy-3-methylglutaryl coenzyme A reductase (2.241 kb), isopentenyl pyrophosphate isomerase (987 bp) and geranyl diphosphate synthase (1.434 kb), were cloned to full-length followed by expression analysis of ten genes vis-à-vis picrosides content analysis. There is maximum accumulation of picrosides in leaf tissue followed by the rhizome and root, and a similar pattern of expression was found in all the ten genes. The genes responded to the modulators of the picrosides biosynthesis. Picrosides accumulation was enhanced by application of hydrogen peroxide and abscisic acid, whereas methyl jasmonate and salicylic acid treatment decreased the content.     

Structure of the GcpE (IspG)-MEcPP complex from Thermus thermophilus

Isoprenoid precursor biosynthesis occurs through the mevalonate or the methylerythritol phosphate (MEP) pathway, used i.e., by humans and by many human pathogens, respectively. In the MEP pathway, 2-C-methyl-d-erythritol-2,4-cyclo-diphosphate (MEcPP) is converted to (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) by the iron-sulfur cluster enzyme HMBPP synthase (GcpE). The presented X-ray structure of the GcpE-MEcPP complex from Thermus thermophilus at 1.55 Å resolution provides valuable information about the catalytic mechanism and for rational inhibitor design. MEcPP binding inside the TIM-barrel funnel induces a 60° rotation of the [4Fe-4S] cluster containing domain onto the TIM-barrel entrance. The apical iron of the [4Fe-4S] cluster ligates with the C3 oxygen atom of MEcPP.     

Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG

In the methylerythritol phosphate pathway for isoprenoid biosynthesis, the GcpE/IspG enzyme catalyzes the conversion of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate. This reaction requires a double one-electron transfer involving a [4Fe-4S] cluster. A thylakoid preparation from spinach chloroplasts was capable in the presence of light to act as sole electron donor for the plant GcpE Arabidopsis thaliana in the absence of any pyridine nucleotide. This is in sharp contrast with the bacterial Escherichia coli GcpE, which requires flavodoxin/flavodoxin reductase and NADPH as reducing system and represents the first proof that the electron flow from photosynthesis can directly act in phototrophic organisms as reducer in the 2-C-methyl-d-erythritol 4-phosphate pathway, most probably via ferredoxin, in the absence of any reducing cofactor. In the dark, the plant GcpE catalysis requires in addition of ferredoxin NADP(+)/ferredoxin oxido-reductase and NADPH as electron shuttle.     

Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: Different role, different evolution

The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Gö1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.     

Isoprenoid biosynthesis in chloroplasts via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) from Arabidopsis thaliana is a [4Fe–4S] protein

The mevalonate-independent methylerythritol phosphate pathway is widespread in bacteria. It is also present in the chloroplasts of all phototrophic organisms. Whereas the first steps, are rather well known, GcpE and LytB, the enzymes catalyzing the last two steps have been much less investigated. 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate is transformed by GcpE into 4-hydroxy-3-methylbut-2-enyl diphosphate, which is converted by LytB into isopentenyl diphosphate or dimethylallyl diphosphate. Only the bacterial GcpE and LytB enzymes have been investigated to some extent, but nothing is known about the corresponding plant enzymes. In this contribution, the prosthetic group of GcpE from the plant Arabidopsis thaliana and the bacterium Escherichia coli has been fully characterized by Mössbauer spectroscopy after reconstitution with ⁵⁷FeCl₃, Na₂S and dithiothreitol. It corresponds to a [4Fe-4S] cluster, suggesting that both plant and bacterial enzymes catalyze the reduction of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate via two consecutive one-electron transfers. In contrast to the bacterial enzyme, which utilizes NADPH/flavodoxin/flavodoxin reductase as a reducing shuttle system, the plant enzyme could not use this reduction system. Enzymatic activity was only detected in the presence of the 5-deazaflavin semiquinone radical.     

The structural basis of chain length control in Rv1086

In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C₅₀) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphate plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan; thus, its synthesis has attracted considerable interest as a potential therapeutic target. Rv1086 is a unique prenyl diphosphate synthase in that it adds only one isoprene unit to geranyl diphosphate, generating the 15-carbon product (E,Z-farnesyl diphosphate). Rv2361c then adds a further seven isoprene units to E,Z-farnesyl diphosphate in a processive manner to generate the 50-carbon prenyl diphosphate, which is then dephosphorylated to generate a carrier for activated sugars. The molecular basis for chain-length discrimination by Rv1086 during synthesis is unknown. We also report the structure of apo Rv1086 with citronellyl diphosphate bound and with the product mimic E,E-farnesyl diphosphate bound. We report the structures of Rv2361c in the apo form, with isopentenyl diphosphate bound and with a substrate analogue, citronellyl diphosphate. The structures confirm the enzymes are very closely related. Detailed comparison reveals structural differences that account for chain-length control in Rv1086. We have tested this hypothesis and have identified a double mutant of Rv1086 that makes a range of longer lipid chains.     

Product chain-length determination mechanism of Z,E-farnesyl diphosphate synthase

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.     

Efficient regeneration for enhanced steviol glycosides production in Stevia rebaudiana (Bertoni)

An efficient method of regeneration for antidiabetic plant (Stevia rebaudiana) has been established for healthy biomass and main steviol glycosides (SGs) production, using different PGRs and agar concentrations. Higher callus induction (93.3%) was recorded when leaf explants were placed on an MS medium supplemented with 3.5 gL⁻¹ agar and 2.0 mgL⁻¹ 2,4-D. The addition of 7.0 gL⁻¹ agar and BA (1.0, 2.0 and 4.0 mgL⁻¹) significantly (P < 0.01) influences shooting response (100%). A maximum mean shoot length (13.03 cm) and 28 shoots per explant were observed on a medium containing 1.0 mgL⁻¹ BA. However, the maximum number of leaves (132.67) was encouraged by the addition of BA (1.0 mgL⁻¹) and Kin (1.0 mgL⁻¹). Lower agar (3.5 gL⁻¹), IAA (2.0 mgL⁻¹), and NAA (2.0 mgL⁻¹) concentrations significantly influence the rooting percent (100%), the mean root length (2.9 cm), and the number of roots per plantlet (26.3). These plantlets were successfully acclimatized in the soil. The BA (3.0 mgL⁻¹) in combination with Kin (3.0 mgL⁻¹) and 3.5 gL⁻¹ agar increases dulcoside-A content (Dul-A; 71.8 μg/g-DW) in shoots compared to control (50.81 μg/g-DW). Similar PGRs with 7.0 gL⁻¹ significantly increases the production of steviosides (Stev. 82.48 μg/g-DW). A higher rebaudioside-A content (Reb-A; 12.35 μg/g-DW) was observed in shoots that underwent the addition of BA (1.0 mgL⁻¹) and 7.0 gL⁻¹ agar than in control (07.39 μg/g-DW). Hereby, we developed an efficient and cost-effective method for regeneration and major SGs production, which could be helpful for future studies on this species.     

Activity of chalcones derived from 2,4,5-trimethoxybenzaldehyde against Meloidogyne exigua and in silico interaction of one chalcone with a putative caffeic acid 3-O-methyltransferase from Meloidogyne incognita

Meloidogyne exigua is a parasitic nematode of plants that causes great losses to coffee farmers. In an effort to develop parasitic controls, 154 chalcones were synthesized and screened for activity against this nematode. The best results were obtained with (2E)-1-(4′-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (6) with a 50% lethal concentration (LC₅₀) of 171 μg/ml against M. exigua second-stage juveniles, in comparison to the commercially-available nematicide carbofuran which had an LC₅₀ of 260 μg/ml under the same conditions. When coffee plants were used, 6 reduced the nematode population to ∼50% of that observed in control plants. To investigate the mechanism of action of 6, an in silico study was carried out, which indicated that 6 may act against M. exigua through inhibition of a putative caffeic acid 3-O-methyltransferase homodimer, the amino acid sequence of which was determined by examining the genome of Meloidogyne incognita.     

Prenylated flavonoids of the leaves of Macaranga conifera with inhibitory activity against cyclooxygenase-2

Two prenylated flavonoid derivatives, 5-hydroxy-4'-methoxy-2",2"-dimethylpyrano-(7,8:6",5")flavanone (1) and 5,4'-dihydroxy-[2"-(1-hydroxy-1-methylethyl)dihydrofurano]-(7,8:5",4")flavanone (2), were isolated from an ethyl acetate-soluble extract of the leaves of Macaranga conifera using an in vitro activity-guided fractionation procedure based on the inhibition of cyclooxygenase-2. Also obtained were eight known compounds, 5,7-dihydroxy-4'-methoxy-8-(3-methylbut-2-enyl)flavanone (3), lonchocarpol A (4), sophoraflavanone B (5), 5,7-dihydroxy-4'-methoxy-8-(2-hydroxy-3-methylbut-3-enyl)flavanone (6), tomentosanol D (7), lupinifolinol (8), isolicoflavonol (9), and 20-epibryonolic acid (10). The structures of compounds 1 and 2 were determined using spectroscopic methods. All isolates were tested for their inhibitory effects against both cyclooxygenases-1 and -2, and selected compounds were evaluated in a mouse mammary organ culture assay.     

Coincident isolation of a novel homoisoflavonoid from Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae)

We report on the first phytochemical investigation of a member of the African genus Resnova (Hyacinthoideae: Hyacinthaceae). From the dichloromethane extract of the bulbs of both Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae) a novel 3-benzyl-4-chromanone homoisoflavonoid, 5,6-dimethoxy-7-hydroxy-3-(4′-hydroxybenzyl)-4-chromanone, was isolated. A further 11 known homoisoflavonoids were also identified, the 12 in total presenting a clear biosynthetic sequence. Eight of the 12 compounds found were common to both species.