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.     

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-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) from Thermus thermophilus

Isoprenoids are biosynthesized via the mevalonate or the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways the latter being used by most pathogenic bacteria, some parasitic protozoa, plant plastids, but not by animals. We determined the X-ray structure of the homodimeric [4Fe–4S] cluster carrying E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) of Thermus thermophilus which catalyzes the penultimate reaction of the MEP pathway and is therefore an attractive target for drug development. The [4Fe–4S] cluster ligated to three cysteines and one glutamate is encapsulated at the intersubunit interface. The substrate binding site lies in front of an (αβ)₈ barrel. The great [4Fe–4S] cluster-substrate distance implicates large-scale domain rearrangements during the reaction cycle.

Structured summary

gcpEbinds to gcpE by x-ray crystallography (View interaction)     

fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis

Although flavodoxin I is indispensable for Escherichia coli growth, the exact pathway(s) where flavodoxin I is essential has not been identified. We performed transposon mutagenesis of the flavodoxin I gene, fldA, in an E. coli strain that expressed mevalonate pathway enzymes and that had a point mutation in the lytB gene of the MEP pathway resulting in the accumulation of (E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP). Disruption of fldA abrogated mevalonate-independent growth and dramatically decreased HMBPP levels. The fldA- mutant grew with mevalonate indicating that the essential role of flavodoxin I under aerobic conditions is in the MEP pathway. Growth was restored by fldA complementation. Since GcpE (which synthesizes HMBPP) and LytB are iron-sulfur enzymes that require a reducing system for their activity, we propose that flavodoxin is essential for GcpE and possibly LytB activity. Thus, the essential role for flavodoxin I in E. coli is in the MEP pathway for isoprenoid biosynthesis.     

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP(+) reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP(+) reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.     

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.     

A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni)

The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.     

Scalable biocatalytic synthesis of optically pure ethyl (R)-2-hydroxy-4-phenylbutyrate using a recombinant E. coli with high catalyst yield

Ethyl (R)-2-hydroxy-4-phenylbutanoate [(R)-HPBE] is a versatile and important chiral intermediate for the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Recombinant E. coli strain coexpressing a novel NADPH-dependent carbonyl reductase gene iolS and glucose dehydrogenase gene gdh from Bacillus subtilis showed excellent catalytic activity in (R)-HPBE production by asymmetric reduction. IolS exhibited high stereoselectivity (>98.5% ee) toward α-ketoesters substrates, whereas fluctuant ee values (53.2–99.5%) for β-ketoesters with different halogen substitution groups. Strategies including aqueous/organic biphasic system and substrate fed-batch were adopted to improve the biocatalytic process. In a 1-L aqueous/octanol biphasic reaction system, (R)-HPBE was produced in 99.5% ee with an exceptional catalyst yield (gproduct/gcatalyst) of 31.7 via bioreduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) at 330 g/L.     

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.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

Reduced glycerol incorporation into phospholipids contributes to impaired intra-erythrocytic growth of glycerol kinase knockout Plasmodium falciparum parasites

Background

Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.

Methods

The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.

Results

Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. ¹⁴C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.

Conclusions

P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.

General significance

These studies provide new insight into glycerolipid metabolism in P. falciparum.     

Interspecific somatic hybrids Solanum villosum (+) S. tuberosum, resistant to Phytophthora infestans

The interspecific somatic hybrids 4x S. villosum (+) 2x S. tuberosum clone DG 81-68 (VT hybrids) were obtained and characterized molecularly and cytogenetically.     

Furanocoumarins from Dorsteniafoetida

The linear furanocoumarins 5-(2,3-epoxy-3-methyl-butoxy)-chalepensin, 5-methoxy-3-(3-methyl-2,3-dihydroxybutyl)-psoralen-diacetate (7), 5-methoxy-3-[3-(β-d-glucopyranosyloxy)-2-acetyloxy-3-methyl-butyl]-psoralen and 5-(3-methyl-2,3-dihydroxybutyloxy)-3-[3-(β-d-glucopyranosyloxy)-2-hydroxy-3-methyl-butyl]-psoralen, and the coumarin derivative 7-hydroxy-5-methoxy-6-carboxymethyl-3-[3-(β-d-glucopyranosyloxy)-2-hydroxy-3-methyl-butyl]-coumarin were isolated from the leaves of Dorstenia foetida (Moraceae) along with the known compounds psoralen, bergapten, isopimpinellin, phellopterin, 5-methoxychalepensin and turbinatocoumarin. Further furanocoumarins were characterized by ESI-MS/MS investigations. The nonpolar extracts of D. foetida exhibit antifungal, antibacterial and cytotoxic activity, however, no anthelminthic activity.     

Pfs 16 pivotal role in Plasmodium falciparum gametocytogenesis: A potential antiplasmodial drug target

Mature gametocytes, the sexual stage of Plasmodium falciparum, ensure the continued transmission of malaria from the human host to the mosquito vector. Even if gametocytes are not implicated in the malaria physiopathology it is crucial to the spread of malaria. Gametocytes are to be a key target for drugs used against Plasmodium in public health. The expression levels of 4 sexual-stage specific genes, Pfs 16, Pfs 25, Pfg 27and S 18S rRNA, during gametocytogenesis of various P. falciparum strains were analyzed by a real time PCR assay. The strains showed different capacities to produce mature gametocytes and in parallel different patterns of sexual gene expression. There was a correlation only between Pfs 16 cDNA overexpression in the first 48 h of the culture and the production of mature gametocytes. Pfs 16 is an early marker of the development of mature gametocytes in cultures and is therefore a potential target for new antimalarial drugs.     

Antiplasmodial activity of (I-3,II-3)-biflavonoids and other constituents from Ormocarpum kirkii

Preliminary screening of a series of medicinal plants, traditionally used in Tanzania, showed an IC₅₀ of 15.6-31.2 μg/ml for the crude extract of the root of Ormocarpum kirkii S. Moore (Papilionaceae) against Plasmodium falciparum. A bioguided isolation was performed in order to isolate the active constituents. Twelve constituents were obtained and identified using NMR and MS data, and optical rotation measurements. The compounds comprised seven (I-3,II-3)-biflavonoids, three (I-3,II-3)-bi-4-phenyldihydrocoumarins, an isoflavanone and a C-glucosylated flavone. Six compounds, liquiritigeninyl-(I-3,II-3)-naringenin, apigeninyl-(I-3,II-3)-naringenin, 7-O-β-D-glucopyranosylchamaejasmin, (3R,4S,3″R,4″S)-5,5″-di-O-methyldiphysin, 7-O-β-D-glucopyranosyldiphysin, and 4″-hydroxydiphysolone, were isolated in addition to six known components. The compounds were evaluated for antimicrobial activity in a broad screening panel, including P. falciparum. Seven of these showed antiplasmodial activity; isochamaejasmin being the most active with an IC₅₀ of 7.3 ± 3.8 μM, but the selectivity was rather limited. Thus, these constituents may contribute, at least in part, to the antimalarial use of O. kirkii in traditional medicine.     

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.     

Plasmodium falciparum: Genetic and immunogenic characterisation of the rhoptry neck protein PfRON4

The Apicomplexan parasites Toxoplasma and Plasmodium, respectively, cause toxoplasmosis and malaria in humans and although they invade different host cells they share largely conserved invasion mechanisms. Plasmodium falciparum merozoite invasion of red blood cells results from a series of co-ordinated events that comprise attachment of the merozoite, its re-orientation, release of the contents of the invasion-related apical organelles (the rhoptries and micronemes) followed by active propulsion of the merozoite into the cell via an actin-myosin motor. During this process, a tight junction between the parasite and red blood cell plasma membranes is formed and recent studies have identified rhoptry neck proteins, including PfRON4, that are specifically associated with the tight junction during invasion. Here, we report the structure of the gene that encodes PfRON4 and its apparent limited diversity amongst geographically diverse P. falciparum isolates. We also report that PfRON4 protein sequences elicit immunogenic responses in natural human malaria infections.     

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.     

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.