Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-¹³C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-¹³C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the ¹³C NMR spectra due to loss of the label as ¹³CO₂. Uptake of [3-¹³C]pyruvate by the cells was considerably slower than with [2-¹³C]acetate; however, the labelling patterns were similar with the exception of increased [3-¹³C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-¹³C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine.     

Biosynthesis of andrographolide in Andrographis paniculata

Andrographolide, a diterpene lactone, is isolated from Andrographis paniculata which is well known for its medicinal properties. The biosynthetic route to andrographolide was studied using [1-¹³C]acetate, [2-¹³C]acetate and [1,6-¹³C₂]glucose. The peak enrichment of eight carbon atoms in the ¹³C NMR spectra of andrographolide suggested that deoxyxylulose pathway (DXP) is the major biosynthetic pathway to this diterpene.The contribution of the mevalonic acid pathway (MVA) is indicated by the observed ¹³C-labeling pattern, and because the labeling patterns indicate a simultaneous contribution of both methyl erythritol phosphate (MEP) and MVA pathways it can be deduced that cross-talk occurs between plastids and cytoplasm.     

Structure and biosynthesis of 5-deoxyfusarubin and anhydro-5-deoxyfusarubin,naphthaquinone pigments from Nectria haematococca

Two naphthaquinone pigments, 5-deoxyfusarubin and anhydro-5-deoxyfusarubin, were isolated from a yellow strain of the fungus Nectria haematococca. The ¹³C NMR spectra of 2 biosynthetically labelled from [1-¹³C]acetate confirmed the structures of the pigments and established the heptaketide origin of the molecules.     

Biosynthesis of artemisinin – revisited

Artemisinin is a well-known antimalarial drug isolated from the Artemisia annua plant. The biosynthesis of this well-known molecule has been reinvestigated by using [1-¹³C]acetate, [2-¹³C]acetate, and [1,6-¹³C₂]glucose. The ¹³C peak enrichment in artemisinin was observed in six and nine carbon atoms from [1-¹³C]acetate and [2-¹³C]acetate, respectively. The ¹³C NMR spectra of ¹³C-enriched artemisinin suggested that the mevalonic acid (MVA) pathway is the predominant route to biosynthesis of this sesquiterpene. On the other hand, the peak enrichment of five carbons of ¹³C-artemisinin including carbon atoms originating from methyls of dimethylallyl group of geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) was observed from [1,6-¹³C₂]glucose. This suggested that GPP which is supposed to be biosynthesized in plastids travels from plastids to cytosol through the plastidial wall and combines with isopentenyl pyrophosphate (IPP) to form the (E,E)-FPP which finally cyclizes and oxidizes to artemisinin. In this way the DXP pathway also contributes to the biosynthesis of this sesquiterpene.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds

Csypyrones B1, B2 and B3 are α-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-¹³C] and [2-¹³C] acetate feeding experiments. ¹³C NMR analyses of the methyl esters of the csypyrone B compounds fed with the ¹³C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus.     

4D Non-uniformly sampled C,C-NOESY experiment for sequential assignment of 13C,15N-labeled RNAs

¹³C spin diluted protein samples can be produced using [1-¹³C] and [2-¹³C]-glucose (Glc) carbon sources in the bacterial growth medium. The ¹³C spin dilution results in favorable ¹³C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-¹³C]- and [2-¹³C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-¹³C]-glycerol (¹³C labeled Cα sites on a ¹²C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17–26, 2011 ). Inspired by this approach and our own recent results using [2-¹³C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of ¹³C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle.     

The structures and biosynthesis of multicolanic,multicolic, and multicolosic acids,novel tetronic acid metabolites of penicillium multicolor

Multicolanic, multicolic, and multicolosic acids, metabolites of Penicillium multicolor, are shown by chemical transformations and spectroscopic methods to be 4-ylidenetetronic acids with structures (I), (II), and (III), respectively. The biosynthesis of these metabolites from acetate, via oxidative fission of preformed 6-pentylresorcylic acid is established by incorporation studies with [1-¹³C]-, [2-¹³C]-, [1,2-¹³C]acetate and ethyl [2-¹⁴C]-6-pentylresorcylate.     

In VivoC NMR Study of the Bidirectional Reactions of the Wood–Werkman Cycle and around the Pyruvate Node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici

This study used in vivo¹³C NMR spectroscopy to directly examine bidirectional reactions of the Wood–Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-¹³C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 μmol min⁻¹ g⁻¹ (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood–Werkman cycle was evident from the formation of intermediates such as [3-¹³C]pyruvate and [3-¹³C]malate and of products like [2-¹³C]acetate from [2-¹³C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-¹³C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.     

Biosynthesis of internally branched monomethylalkanes in the cockroach Periplaneta fuliginosa.

Sodium [1-¹⁴C]acetate, sodium [1-¹⁴C]propionate, sodium [2-¹⁴C]propionate, sodium [3-¹⁴C]propionate and sodium [methyl-¹⁴C]methylmalonate were readily incorporated into the cuticular hydrocarbons of nymphal stages of the cockroach Periplaneta fuliginosa both in vivo and in vitro, whereas no incorporation of [methyl-¹⁴C]methionine was observed. The alkanes of the nymphal stages of this insect are 25+% n-alkanes, 14% 3-methylalkanes, and 59+% internally branched monomethylalkanes, principally 13-methylpentacosane. Sodium [1-¹⁴C]acetate was incorporated into each class of alkane at about its percentage composition. In contrast, labeled sodium propionate and sodium methylmalonate were preferentially incorporated into the branched fractions. Radio-gas-liquid chromatography showed that sodium [1-¹⁴C]propionate was incorporated almost exclusively into 3-methyltricosane and 13-methylpentacosane, whereas sodium [1-¹⁴C]acetate was incorporated into each glc peak at about its percentage composition. These data suggest that propionate, incorporated during chain elongation, serves as the branching methyl group donor for both the 3-methyl and the internally branched monomethylalkanes in insects. The location of hydrocarbon synthesis in P. fuliginosa was studied using an in vitro tissue slice system. Excised cuticle slices, with adhering fat body tissue removed, gave good incorporation of labeled substrates into the hydrocarbon fraction. No hydrocarbon synthesis was observed in fat body preparations.     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed C NMR

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the ¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting ¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴-10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However, ¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl ¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, ¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

Production of Specifically Labeled Compounds by Methanobacterium espanolae Grown on H2-CO2 plus [13C]Acetate

Methanobacterium espanolae, an acidiphilic methanogen, required acetate for maximal growth on H₂-CO₂. In the presence of 5 to 15 mM acetate, at a growth pH of 5.5, the μ_max was 0.05 h^-1. M. espanolae consumed 12.3 mM acetate during 96 h of incubation at 35°C with shaking at 100 rpm. At initial acetate levels of 2.5 to 10.0 mM, the amount of biomass produced was dependent on the amount of acetate in the medium. ¹³C nuclear magnetic resonance spectra of protein hydrolysates obtained from cultures grown on [1-¹³C]- or [2-¹³C]acetate indicated that an incomplete tricarboxylic acid pathway, operating in the reductive direction, was functional in this methanogen. The amino acids were labeled with a very high degree of specificity and at greater than 90% enrichment levels. Less than 2% label randomization occurred between positions primarily labeled from either the carboxyl or methyl group of acetate, and very little label was transferred to positions primarily labeled from CO₂. The labeling pattern of carbohydrates was typical for glucogenesis from pyruvate. This methanogen, by virtue of the properties described above and its ability to incorporate all of the available acetate (10 mM or lower) from the growth medium, has advantages over other microorganisms for use in the production of specifically labeled compounds.     

A new biosynthetic pathway of porphyrins from isopropanol

A new biosynthetic pathway, which can produce both vitamin B₁₂ and large amounts of porphyrins from isopropanol, was identified in Arthrobacter hyalinus using carbon-13 stable isotope tracer techniques and carbon-13 nuclear magnetic resonance (¹³C-NMR) spectroscopy. Studies on the incorporation of [2-¹³C]isopropanol, [1- or 2-¹³C]sodium acetate, l-[1-¹³C]glutamate, and [1-, 2-, 3-, 4-, 5-¹³C]5-aminolevulinic acid into uroporphyrinogen III showed that isopropanol was metabolized into uroporphyrinogen III through acetyl CoA and that 5-aminolevulinic acid was produced from l-glutamic acid and not via Shemin's pathway.     

The biosynthesis of trichothecin from acetate- [1,2- 13C2]

The coupling pattern of trichothecin biosynthesized from acetate-[1,2-¹³C₂] is in accord with previous enrichment studies. Multiple labelling was observed. Exogenous acetate has been shown to inhibit the utilization of glucose and the incorporation of radioactivity from pyruvate-[2-¹⁴C] and citrate-[1,5-¹⁴C] into the metabolites. Two pairs of ¹³C NMR assignments are interchanged.     

Residue-specific millisecond to microsecond fluctuations in bacteriorhodopsin induced by disrupted or disorganized two-dimensional crystalline lattice, through modified lipid-helix and helix-helix interactions, as revealed by C NMR

We have recorded ¹³C NMR spectra of [3-¹³C]-, [1-¹³C]Ala-, and [1-¹³C]Val-labeled bacteriorhodopsin (bR), W80L and W12L mutants and bacterio-opsin (bO) from retinal-deficient E1001 strain, in order to examine the possibility of their millisecond to microsecond local fluctuations with correlation time in the order of 10⁻⁴ to 10⁻⁵ s, induced or prevented by disruption or assembly of two-dimensional (2D) crystalline lattice, respectively, at ambient temperature. The presence of disrupted or disorganized 2D lattice for W12L, W80L and bO from E1001 strain was readily visualized by increased relative proportions of surrounding lipids per protein, together with their broadened ¹³C NMR signals of transmembrane α-helices and loops in [3-¹³C]Ala-labeled proteins, with reference to those of wild-type. In contrast, ¹³C CP-MAS NMR spectra of [1-¹³C]Ala- and Val-labeled these mutants were almost completely suppressed, owing to the presence of fluctuations with time scale of 10⁻⁴ s interfered with magic angle spinning. In particular, ¹³C NMR signals of [1-¹³C]Ala-labeled transmembrane α-helices of wild-type were almost completely suppressed at the interface between the surface and inner part (up to 8.7 Å deep from the surface) with reference to those of the similarly suppressed peaks by Mn²⁺-induced accelerated spin-spin relaxation rate. Such fluctuation-induced suppression of ¹³C NMR peaks from the interfacial regions, however, was less significant for [1-¹³C]Val-labeled proteins, because fluctuation motions in Val residues with bulky side-chains at the C_α moiety were modified to those of longer correlation time (>10⁻⁴ s), if any, by residue-specific manner. To support this view, we found that such suppressed ¹³C NMR signals of [1-¹³C]Ala-labeled peaks in the wild-type were recovered for D85N and bO in which correlation times of fluctuations were shifted to the order of 10⁻⁵ s due to modified helix-helix interactions as previously pointed out [Biochemistry, 39 (2000) 14472; J. Biochem. (Tokyo) 127 (2000) 861].     

Backbone dynamics of membrane proteins in lipid bilayers: the effect of two-dimensional array formation as revealed by site-directed solid-state C NMR studies on [3-C]Ala- and [1-C]Val-labeled bacteriorhodopsin

We have recorded site-directed solid-state ¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled bacteriorhodopsin (bR) as a typical membrane protein in lipid bilayers, to examine the effect of formation of two-dimensional (2D) lattice or array of the proteins toward backbone dynamics, to search the optimum condition to be able to record full ¹³C NMR signals from whole area of proteins. Well-resolved ¹³C NMR signals were recorded for monomeric [3-¹³C]Ala-bR in egg phosphatidylcholine (PC) bilayer at ambient temperature, although several ¹³C NMR signals from the loops and transmembrane α-helices were still suppressed. This is because monomeric bR reconstituted into egg PC, dimyristoylphosphatidylcholine (DMPC) or dipalmytoylphosphatidylcholine (DPPC) bilayers undergoes conformational fluctuations with frequency in the order of 10⁴-10⁵ Hz at ambient temperature, which is interfered with frequency of magic angle spinning or proton decoupling. It turned out, however, that the ¹³C NMR signals of purple membrane (PM) were almost fully recovered in gel phase lipids of DMPC or DPPC bilayers at around 0 °C. This finding is interpreted in terms of aggregation of bR in DMPC or DPPC bilayers to 2D hexagonal array in the presence of endogenous lipids at low temperature, resulting in favorable backbone dynamics for ¹³C NMR observation. It is therefore concluded that [3-¹³C]Ala-bR reconstituted in egg PC, DMPC or DPPC bilayers at ambient temperature, or [3-¹³C]Ala- and [1-¹³C]Val-bR at low temperature gave rise to well-resolved ¹³C NMR signals, although they are not always completely the same as those of 2D hexagonal lattice from PM.     

Biosynthesis of 4-methyl-1-nonanol: Female-produced sex pheromone of the yellow mealworm beetle,Tenebrio molitor (Coleoptera: Tenebrionidae)

The objective of this study was to elucidate the biosynthetic route to 4-methyl-1-nonanol, the female-produced sex pheromone of the yellow mealworm beetle, Tenebrio molitor L. The biosynthetic route to the pheromone was examined by (i) allowing the females to feed on defatted bran coated with a stable isotope-labeled putative precursor ([1-¹³C]acetate, [1-¹³C]propionate, [1-¹³C]pentanoate, [1-¹³C]2-methylheptanoic acid, or [²H₂]4-methylnonanoic acid); (ii) determining if the precursors were incorporated by analyzing the emitted pheromone by gas chromatography/selected ion monitoring-mass spectroscopy (GC/SIM-MS); (iii) where the pheromone was isotopically-enriched, determining the position of the isotopic label(s) through comparison of the MS fragmentation pattern with that of unlabelled 4-methyl-1-nonanol. Although the incorporation of [1-¹³C]acetate into 4-methyl-1-nonanol could not be detected, relatively large proportions of the pheromone were produced from the other precursors tested: 81% from [²H₂]4-methylnonanoic acid, 45% from [1-¹³C]2-methylheptanoic acid, 16% from [1-¹³C]pentanoate, and 35% from [1-¹³C]propionate (27% from only one unit, and 7.8% from two units). The results indicate that 4-methyl-1-nonanol is produced through a modification of normal fatty acid biosynthesis: initiation of the pathway with one unit of propionate results in the uneven number of carbons in the chain; incorporation of another unit of propionate during elongation provides the methyl branch; reduction of 4-methylnonanoic acid produces the alcohol pheromone. The elucidation of the biosynthetic pathway of 4-methyl-1-nonanol biosynthesis in the yellow mealworm is the first step towards understanding the biochemistry of sex pheromone production in this species.     

The biosynthesis of the antioxidant caffeic acid derivative subulatin by the liverwort Jungermannia subulata cultured in vitro

The in vitro cultured liverwort Jungermannia subulata produces the unique molecule subulatin. In this study, we examined the incorporation of [1-¹³C] and [1,2-¹³C₂] glucose, [2-¹³C] arabinose, [2-¹³C] caffeic acid, and [1-¹³C] phenylalanine into subulatin. The trilobatinoic acid C unit of subulatin incorporated ¹³C atoms from [1-¹³C] and [1,2-¹³C₂] glucose and from [2-¹³C] arabinose but not from any other of the other precursors. Based on these results and labeling patterns, the trilobatinoic acid C unit of subulatin appears to be biosynthesized from arabinose-5-phosphate and phosphoenolpyruvate.     

Cortical metabolism in pyruvate dehydrogenase deficiency revealed by ex vivo multiplet C NMR of the adult mouse brain

The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the ¹³C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-¹³C₂]glucose (oxidized by both neurons and glia) and [1,2-¹³C₂]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by ¹³C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of ¹³C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The ¹³C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-¹³C]acetate relative to [1,6-¹³C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron–glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.