Incorporation of [1-(13)C]1-deoxy-D-xylulose into isoprenoids of the liverwort Conocephalum conicum

The incorporation of (13)C labeled 1-deoxy-D-xylulose into the monoterpene bornyl acetate, the sesquiterpene cubebanol, and the diterpene phytol has been studied in axenic cultures of the liverwort Conocephalum conicum. Quantitative (13)C NMR spectroscopic analysis of the labeling patterns of the sesquiterpene indicated a possible degradation of 1-deoxy-D-xylulose to acetate and subsequent incorporation via the mevalonic acid pathway. In bornyl acetate, the labeling occurred only in the acetate moiety whereas the isoprene units remained unlabelled. The isoprene units of the diterpene phytol showed incorporation of intact deoxy-D-xylulose. These results indicate the involvement of both IPP biosynthetic pathways and two independently operating compartments/cell types with MEP pathway machinery. One MEP compartment is presumably the plastid where phytol is formed; the second, involved in the build-up of the isoprene part of bornyl acetate, might be located in the oil cells. The acetylation of borneol to bornyl acetate in turn occurs in a cellular compartment that is not involved in the build-up of the isoprene units of borneol.     

Biosynthesis of anthraquinones in cell cultures of Cinchona 'Robusta' proceeds via the methylerythritol 4-phosphate pathway.

Robustaquinone B was found as a major anthraquinone in cell cultures of Cinchona 'Robusta' after treatment with a fungal elicitor. Anthraquinones in Cinchona are considered to be of the Rubia type, i.e. rings A and B are derived from chorismate and alpha-ketoglutarate, whereas ring C is formed from isopentenyl diphosphate (IPP). To determine the origin of IPP, either formed via the mevalonic acid pathway or the 2-C-methyl-D-erythritol 4-phosphate pathway, the incorporation of [1-13C]glucose into robustaquinone B was studied. The 13C labeling of robustaquinone B was analyzed by one- and two-dimensional NMR spectroscopy and the labeling pattern was compared with the hypothetical labeling patterns obtained via the different biosynthetic pathways. The results clearly show that the IPP, constituting the ring C of robustaquinone B, is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. Moreover, the data also confirm that rings A and B of robustaquinone B are formed from chorismate and alpha-ketoglutarate via o-succinylbenzoate.     

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.     

Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis: unidirectional transport of intermediates across the chloroplast envelope membrane

In higher plants, two independent pathways are responsible for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central five-carbon precursors of all isoprenoids. The cytosolic pathway, which involves mevalonate (MVA) as a key intermediate, provides the precursor molecules for sterols, ubiquinone, and certain sesquiterpenes, whereas the plastidial MVA-independent pathway is involved in the formation of precursors for the biosynthesis of isoprene, monoterpenes, diterpenes, carotenoids, abscisic acid, and the side chains of chlorophylls, tocopherols, and plastoquinone. Recent experiments provided indirect evidence for the presence of an export system for isoprenoid intermediates from the plastids to the cytosol in Arabidopsis thaliana. Here we report that isolated chloroplasts (from spinach, kale, and Indian mustard), envelope membrane vesicles, and proteoliposomes prepared from the solubilized proteins of envelope membranes (from spinach) are capable of the efficient transport of isopentenyl diphosphate and geranyl diphosphate. Lower rates of transport were observed with the substrates farnesyl diphosphate and dimethylallyl diphosphate, whereas geranylgeranyl diphosphate and mevalonate were not transported with appreciable efficiency. Our data suggest that plastid membranes possess a unidirectional proton symport system for the export of specific isoprenoid intermediates involved in the metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis.     

Optimized enzymatic preparation of 1-deoxy-d-xylulose 5-phosphate

The preparation of 1-deoxy-d-xylulose 5-phosphate, the key intermediate of MEP biosynthetic pathway for terpenoids by using recombinant 1-deoxy-d-xylulose 5-phosphate synthase of Rhodobacter capsulatus was optimized. The simple one-pot synthesis coupling with a newly established ion-exchange purification process affords the target compound with more than 80% yield and high purity (>95%). The procedure can also be employed to synthesize isotope labeled 1-deoxy-d-xylulose 5-phosphate by using isotope labeled starting materials.     

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.     

A colorimetric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase activity

A new method for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase, the enzyme catalyzing the third reaction of the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesis of isoprenoids, is described. This is an end-point assay based on the transformation of inorganic pyrophosphate, one of the products of the reaction, to phosphate by using inorganic pyrophosphatase as auxiliary enzyme. The phosphate formed is reacted then with the dye malachite green to yield a colored product which can be determined spectrophotometrically. The method is easy to perform, sensitive, and robust and can be used in automated high-throughput screening analyses for the search of inhibitors of the enzyme.     

Possible direct involvement of the active-site [4Fe-4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

The GcpE enzyme converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the penultimate step of the DOXP pathway for isoprene biosynthesis. Purification of the enzyme under exclusion of air leads to a preparation that contains solely [4Fe-4S] clusters. Kinetic studies showed that in the presence of the artificial reductant dithionite and MEcPP a new transient iron-sulfur-based signal is detected in electron paramagnetic resonance (EPR) spectroscopy. Similarity of this EPR signal to that detected in ferredoxin:thioredoxin reductase indicates that during the reaction an intermediate is directly bound to the active-site cluster.     

Cloning and in vitro characterization of dTDP-6-deoxy-l-talose biosynthetic genes from Kitasatospora kifunensis featuring the dTDP-6-deoxy-l-lyxo-4-hexulose reductase that synthesizes dTDP-6-deoxy-l-talose

Kitasatospora kifunensis, the talosin producer, was used as a source for the dTDP-6-deoxy-l-talose (dTDP-6dTal) biosynthetic gene cluster, serving as a template for four recombinant proteins of RmlA_Kkf, RmlB_Kkf, RmlC_Kkf, and Tal, which complete the biosynthesis of dTDP-6dTal from dTTP, α-d-glucose-1-phosphate, and NAD(P)H. The identity of dTDP-6dTal was validated using ¹H and ¹³C NMR spectroscopy. K. kifunensistal and tll, the known dTDP-6dTal synthase gene of Actinobacillus actinomycetemcomitans origin, have low sequence similarity and are distantly related within the NDP-6-deoxy-4-ketohexose reductase family, providing an example of the genetic diversity within the dTDP-6dTal biosynthetic pathway.     

Theoretical study of the experimental coordination behavior of N-(2-aminophenyl)-D-glycero-D-gulo-heptonamide to Hg(II) ion

The reactivity of N-(2-aminophenyl)-d-glycero-d-gulo-heptonamide (adgha), with the group 12 cations, Zn(II), Cd(II), and Hg(II), was studied in DMSO-d₆ solution. The studied system showed a selective coordination to Hg(II), and the products formed were characterized by ¹H and ¹³C NMR in DMSO-d₆ solution and fast atom bombardment (FAB⁺) mass spectra. The expected coordination compounds, [Hg(adgha)](NO₃)₂ and [Hg(adgha)₂](NO₃)₂, were observed as unstable intermediates that decompose to bis-[2-(d-glycero-d-gulo-hexahydroxyhexyl)-benzimidazole-κN]mercury(II) dinitrate, [Hg(ghbz)₂](NO₃)₂. The chemical transformation of the complexes was followed by NMR experiments, and the nature of the species formed is sustained by a theoretical study done using DFT methodology. From this study, we propose the structure of the complexes formed in solution, the relative stability of the species formed, and the possible role of the solvent in the observed transformations.     

Biosynthesis of pyrethrin I in seedlings of Chrysanthemum cinerariaefolium

The biosynthetic pathway to natural pyrethrins in Chrysanthemum cinerariaefolium seedlings was studied using [1-13C]d-glucose as a precursor, with pyrethrin I isolated using HPLC from a leaf extract. The 13C NMR spectrum of pyrethrin I from the precursor-administered seedlings indicated that the acid moiety was biosynthesized from d-glucose via 2-C-methyl-d-erythritol 4-phosphate, whereas the alcohol moiety was possibly biosynthesized from linolenic acid.     

Oxidation of methyl α-d-galactopyranoside by galactose oxidase: products formed and optimization of reaction conditions for production of aldehyde

The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl α-d-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by ¹H NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl α-d-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 °C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl α-d-galactopyranosiduronic acid) and an α,β-unsaturated aldehyde (methyl 4-deoxy-α-d-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. ¹H and ¹³C NMR data of the products are reported for the α,β-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d₆ for all the products for the first time. Oxidation of d-raffinose (α-d-galactopyranosyl-(1-6)-α-d-glucopyranosyl-(1-2)-β-d-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde.     

Synthesis of new N-substituted 3,4,5-trihydroxypiperidin-2-ones from d-ribono-1,4-lactone

d-Ribono-1,4-lactone was treated with ethylamine in DMF to afford N-ethyl-d-ribonamide 8a in quantitative yield. Using this reaction procedure, N-butyl, N-hexyl, N-dodecyl, N-benzyl, N-(3-methyl-pyridinyl)-, N-(2-hydroxy-ethyl)-, and N-(2-cyano-ethyl)-d-ribonamides 8b-h were obtained in quantitative yield. Bromination of the amides 8a-e with acetyl bromide in dioxane followed by acetylation gave 2,3,4-tri-O-acetyl-5-bromo-5-deoxy-N-ethyl, N-butyl, N-hexyl, N-dodecyl, and N-benzyl-d-ribonamides 9a-e in 40-54% yields. To obtain 2,3,4-tri-O-acetyl-5-bromo-5-deoxy-N-(3-methyl-pyridinyl)-, N-(2-hydroxy-ethyl)-, and N-(2-cyano-ethyl)-9f-h, the bromination is necessary before the amidation reaction. Treatment of the bromoamides 9a-h with NaH in DMF followed by methanolysis affords N-alkyl-d-ribono-1,5-lactams 12a-h in quantitative yield.     

New evidence for cofactor's amino group function in thiamin catalysis by transketolase

Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4′-methylamino-thiamin diphosphate. We demonstrated the kinetic stability of the dihydroxyethyl carbanion/enamine intermediate to be dependent on the functionality of the 4′-aminopyrimidine moiety of thiamin diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. König, R. Kluger, G.A. Kochetov, G. Schneider, G. Hübner, Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisae, FEBS J. (2005) 272 1326-1342]. This paper extends these investigations of the function of the coenzyme’s aminopyrimidine in transketolase catalysis exemplified for the 4′-monomethylamino-thiamin diphosphate analogue. Here, we report near UV circular dichroism data and NMR-based analysis of reaction intermediates that give evidence for a strong destabilisation of the carbanion/enamine of DHE-4’-monomethylamino-thiamin diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to the conjugate acid of the carbanion/enamine intermediate, an assignment additionally corroborated by ¹H NMR-based intermediate analysis. As opposed to the kinetically stabilized carbanion/enamine intermediate in transketolase when reconstituted with the native cofactor, DHE-4′-monomethylamino-thiamin diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale.     

Inverting character of family GH115 α-glucuronidases

α-Glucuronidases of glycoside hydrolase family 115 of the xylose-fermenting yeast Pichia stipitis and wood-destroying fungus Schizophyllum commune liberate 4-O-methyl-d-glucuronic acid residues from aldouronic acids and glucuronoxylan. The specific activities of both enzymes depended on polymerization degree of the acidic xylooligosaccharides and were inhibited by linear β-1,4-xylooligosaccharides. These results suggest interaction of the enzyme with several xylopyranosyl residues of the xylan main chain. Using ¹H NMR spectroscopy and reduced aldopentaouronic acid (MeGlcA³Xyl₄-ol) as a substrate, it was found that both enzymes are inverting glycoside hydrolases releasing 4-O-methyl-d-glucuronic acid (MeGlcA) as its β-anomer.     

A mutant pyruvate dehydrogenase E1 subunit allows survival of Escherichia coli strains defective in 1-deoxy-D-xylulose 5-phosphate synthase

The 2-C-methyl-D-erythritol 4-phosphate pathway has been proposed as a promising target to develop new antimicrobial agents. However, spontaneous mutations in Escherichia coli were observed to rescue the otherwise lethal loss of the first two enzymes of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), with a relatively high frequency. A mutation in the gene encoding the E1 subunit of the pyruvate dehydrogenase complex was shown to be sufficient to rescue the lack of DXS but not DXR in vivo, suggesting that the mutant enzyme likely allows the synthesis of DXP or an alternative substrate for DXR.     

Feeding of [5,5-2H(2)]-1-desoxy-D-xylulose and [4,4,6,6,6-2H(5)]-mevalolactone to a geosmin-producing Streptomyces sp. and Fossombronia pusilla

The biosynthesis of the trisnor sesquiterpenoid geosmin (4,8a-dimethyl-octahydro-naphthalen-4a-ol) (1) was investigated by feeding labeled [5,5-2H(2)]-1-desoxy-D-xylulose (11), [4,4,6,6,6-(2)H(5)]-mevalolactone (7) and [2,2-2H(2)]-mevalolactone (9) to Streptomyces sp. JP95 and the liverwort Fossombronia pusilla. The micro-organism produced geosmin via the 1-desoxy-D-xylulose pathway, whereas the liverwort exclusively utilized mevalolactone for terpenoid biosynthesis. Analysis of the labeling pattern in the resulting isotopomers of geosmin (1) by mass spectroscopy (EI/MS) revealed that geosmin is synthesized in both organisms by cyclization of farnesyl diphosphate to a germacradiene-type intermediate 4. Further transformations en route to geosmin (1) involve an oxidative dealkylation of an i-propyl substituent, 1,2-reduction of a resulting conjugated diene, and bicyclization of a germacatriene intermediate 13. The transformations largely resemble the biosynthesis of dehydrogeosmin (2) in cactus flowers but differ with respect to the regioselectivity of the side chain dealkylation and 1,2-reduction