Enzymatic preparation of [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol and hydroxypyruvate using the methanol-assimilating system of methylotrophic yeasts

Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C₁-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO₂. When [¹³C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, ¹³C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the ¹³C content of each carbon (atoms/100 atoms) was estimated to be 50%. [¹³C]Methanol was also useful for the enrichment of dihydroxyacetone with ¹³C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of ¹³C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-¹³C]dihydroxyacetone phosphate from [¹³C]methanol; the molar yield of the ester relative to methanol added was 92.5%     

1H, 15N, 13C resonance assignment of the AlgE6R1 subunit from the Azotobacter vinelandii mannuronan C5-epimerase

Isotopically labelled, ¹³C/¹⁵N from of recombinant subunit of the first R-module from alginate C5-epimerase 6 (AlgE6R1) from Azotobacter vinelandii mannuronan C5-epimerase was produced. We report here the ¹H, ¹⁵N, ¹³C resonance assignment of this subunit from AlgE6 epimerase.     

1H, 13C, and 15N NMR assignments of the actinoporin Sticholysin I

Sticholysin I is an actinoporin, a pore forming toxin, of 176 aminoacids produced by the sea anemone Stichodactyla heliantus. Isotopically labelled ¹³C/¹⁵N recombinant protein was produced in E. coli. Here we report the complete NMR ¹⁵N, ¹³C and ¹H chemical shifts assignments of Stn I at pH 4.0 and 25°C (BMRB No. 15927).     

Synthesis of a 13C-Labeled Tracer for Stream DOC: Labeling Tulip Poplar Carbon with 13CO2

Ecosystem tracer-level additions would benefit from a stable isotope-labeled source of complex organic molecules. We tested a method to label tree C with ¹³C and create a stable isotope tracer for stream dissolved organic carbon (DOC) using tulip poplar (Liriodendron tulipifera L.) seedlings. In 2000, seedlings were grown with 0.82 moles of ¹³CO₂ to assess the distribution and level of ¹³C enrichment in the tree tissues. In 2001, seedlings were grown with 25 times more ¹³CO₂ to generate tissues with a ¹³C signal strong enough for a ¹³C-DOC stream tracer addition. ¹³C enrichment in the trees varied in each year and by tissue age and type. Tissues formed during labeling (new) were more enriched in ¹³C than tissues established prior to the ¹³CO₂ injection (old). Stems were most enriched in ¹³C in both new and old tissues. A higher percentage of ¹³CO₂ was incorporated into seedlings in 2000 (59% ±1) than 2001 (43% ±0). Percent ¹³C incorporation among tree tissue types paralleled biomass distributions. Although tree C and ¹³C were equally soluble in both years, a greater percentage of tree C went into solution in 2001 (30%) than 2000 (20%). The water-soluble tree C accounted for approximately 12% of the injected ¹³CO₂ and had both humic and polysaccharide components. Results from a whole-stream ¹³C-DOC tracer addition demonstrated that tree C could be sufficiently labeled with ¹³CO₂ to create a stream DOC isotope tracer with some polymeric constituents.     

Effects of sampling method on foliar δ13C of Leymus chinensis at different scales

Stable carbon isotope composition (δ¹³C) usually shows a negative relationship with precipitation at a large scale. We hypothesized that sampling method affects foliar δ¹³C and its response pattern to precipitation. We selected 11 sites along a precipitation gradient in Inner Mongolia and collected leaves of Leymus chinensis with five or six replications repeatedly in each site from 2009 to 2011. Additionally, we collected leaves of L. chinensis separately from two types of grassland (grazed and fenced) in 2011. Foliar δ¹³C values of all samples were measured. We compared the patterns that foliar δ¹³C to precipitation among different years or different sample sizes, the differences of foliar δ¹³C between grazed and fenced grassland. Whether actual annual precipitation (AAP) or mean annual precipitation (MAP), it was strongly correlated with foliar δ¹³C every year. Significant difference was found between the slopes of foliar δ¹³C to AAP and MAP every year, among the slopes of foliar δ¹³C to AAP from 2009 to 2011. The more samples used at each site the lower and convergent P‐values of the linear regression test between foliar δ¹³C and precipitation. Furthermore, there was significant lower foliar δ¹³C value in presence of grazed type than fenced type grassland. These findings provide evidence that there is significant effect of sampling method to foliar δ¹³C and its response pattern to precipitation of L. chinensis. Our results have valuable implications in methodology for future field sampling studies.     

Discrimination between12C and13C by marine plants

Summary The natural abundance¹³C/¹²C ratios (as δ¹³C) of organic matter of marine macroalgae from Fife and Angus (East Scotland) were measured for comparison with the species' ability to use CO₂ and HCO ₃ ^- for photosynthesis, as deduced from previously published pH-drift measurements. There was a clear difference in δ¹³C values for species able or unable to use HCO ₃ ^- . Six species of Chlorophyta, 12 species of Phaeophyta and 8 species of Rhodophyta that the pH-drift data suggested could use HCO ₃ ^- had δ¹³C values in the range -8.81‰ to -22.55‰. A further 6 species of Rhodophyta which the pH-drift data suggested could only use CO₂ had δ¹³C values in the range -29.90‰ to-34.51‰. One of these six species (Lomentaria articulata) is intertidal; the other five are subtidal and so have no access to atmospheric CO₂ to complicate the analysis. For these species, calculations based on the measured δ¹³C of the algae, the δ¹³C of CO₂ in seawater, and the known¹³C/¹²C discrimination of CO₂ diffusion and RUBISCO carboxylation suggest that only 15–21% of the limitation to photosynthesisin situ results from CO₂ diffusion from the bulk medium to the plastids; the remaining 79–85% is associated with carboxylation reactions (and, via feedback effects, down-stream processes). This analysis has been extended for one of these five species,Delesseria sanguinea, by incorporating data onin situ specific growth rates, respiratory rates measured in the laboratory, and applying Fick's law of diffusion to calculate a boundary layer thickness of 17–24 μm. This value is reasonable for aDelesseria sanguinea frondin situ. For HCO ₃ ^- -using marine macroalgae the range of δ¹³C values measured can be accommodated by a CO₂ efflux from algal cells which range from 0.306 of the gross HCO ₃ ^- influx forEnteromorpha intestinalis (δ¹³C=-8.81‰) in a rockpool to 0.787 forChondrus crispus (δ¹³C=-22.55‰). The relatively high computed CO₂ efflux for those HCO ₃ ^- -users with the more negative δ¹³C values implies a relatively high photon cost of C assimilation; the observed photon costs can be accommodated by assuming coupled, energy-independent inorganic carbon influx and efflux. The observed δ¹³C values are also interpreted in terms of water movement regimes and obtaining CO₂ from the atmosphere. Published δ¹³C values for freshwater macrophytes were compared with the ability of the species to use CO₂ and HCO ₃ ^- and again there was an apparent separation in δ¹³C values for these two groups. δ¹³C values obtained for marine macroalgae for which no pH-drift data are available permit predictions, as yet untested, as to whether they use predominantly CO₂ or HCO ₃ ^-     

Hydrologic linkages between a climate oscillation,river flows,growth, and wood Δ13C of male and female cottonwood trees

To investigate climatic influence on floodplain trees, we analysed interannual correspondences between the Pacific Decadal Oscillation (PDO), river and groundwater hydrology, and growth and wood ¹³C discrimination (Δ¹³C) of narrowleaf cottonwoods (Populus angustifolia) in a semi‐arid prairie region. From the Rocky Mountain headwaters, river discharge (Q) was coordinated with the PDO (1910–2008: r² = 0.46); this pattern extended to the prairie and was amplified by water withdrawal for irrigation. Floodplain groundwater depth was correlated with river stage (r² = 0.96), and the cottonwood trunk basal area growth was coordinated with current‐ and prior‐year Q (1992–2008: r² = 0.51), increasing in the mid‐1990s, and decreasing in 2000 and 2001. Annual Δ¹³C decreased during low‐flow years, especially in trees that were higher or further from the river, suggesting drought stress and stomatal closure, and male trees were more responsive than females (?0.86 versus ?0.43‰). With subsequently increased flows, Δ¹³C increased and growth recovered. This demonstrated the linkages between hydroclimatic variation and cottonwood ecophysiology, and we conclude that cottonwoods will be vulnerable to drought from declining river flows due to water withdrawal and climate change. Trees further from the river could be especially affected, leading to narrowing of floodplain forests along some rivers.     

1H, 13C and 15N resonances of the AlgE62 subunit from Azotobacter vinelandii mannuronan C5-epimerase

The 17.7 kDa R2 module from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been isotopically labeled (¹³C,¹⁵N) and recombinantly expressed. Here we report the ¹H, ¹³C, ¹⁵N resonance assignment of AlgE6R2.     

NMR assignments of 1H, 13C and 15N spectra of methionine sulfoxide reductase B1 from Mus musculus

Isotopically labeled, ¹⁵N and ¹⁵N/¹³C forms of recombinant methionine-r-sulfoxide reductase 1 (MsrB1, SelR) from Mus musculus were produced, in which catalytic selenocysteine was replaced with cysteine. We report here the ¹H, ¹⁵N and ¹³C NMR assignment of the reduced form of this mammalian protein.     

Relationship between δ13C of chironomid remains and methane flux in Swedish lakes

1. Methanogenic carbon can be incorporated by methane‐oxidising bacteria, leading to a ¹³C‐depleted stable carbon isotopic composition (δ¹³C) of chironomids that feed on these microorganisms. This has been shown for the chironomid tribe Chironomini, but very little information is available about the δ¹³C of other abundant chironomid groups and the relationship between chironomid δ¹³C and methane production in lakes. 2. Methane flux was measured at the water surface of seven lakes in Sweden. Furthermore, fluxes from the sediments to the water column were measured in transects in two of the lakes. Methane fluxes were then compared with δ¹³C of chitinous chironomid remains isolated from the lake surface sediments. Several different chironomid groups were examined (Chironomini, Orthocladiinae, Tanypodinae and Tanytarsini). 3. Remains of Orthocladiinae in the seven study lakes had the highest δ¹³C values (?31.3 to ?27.0‰), most likely reflecting δ¹³C of algae and other plant‐derived organic matter. Remains of Chironomini and Tanypodinae had lower δ¹³C values (?33.2 to ?27.6‰ and ?33.6 to ?28.0‰, respectively). A significant negative correlation was observed between methane fluxes at the lake surface and δ¹³C of Chironomini (r = ?0.90, P = 0.006). Methane release from the sediments was also negatively correlated with δ¹³C of Chironomini (r = ?0.67, P = 0.025) in the transect samples obtained from two of the lakes. The remains of other chironomid taxa were only weakly or not correlated with methane fluxes measured in our study lakes (P > 0.05). 4. Selective incorporation of methane‐derived carbon can explain the observed correlations between methane fluxes and δ¹³C values of Chironomini. Remains of this group might therefore have the potential to provide information about past changes in methane availability in lakes using sediment records. However, differences in productivity, algal δ¹³C composition and the importance of allochthonous organic matter input between the studied lakes may also have influenced Chironomini δ¹³C. More detailed studies with a higher number of analysed samples and detailed measurement of δ¹³C of different ecosystem components (e.g. methane, dissolved inorganic carbon) will be necessary to further resolve the relative contribution of different carbon sources to δ¹³C of chironomid remains.     

Measurement of methyl 13C-1H cross-correlation in uniformly 13C-, 15N-, labeled proteins

An understanding of side chain motions in protein is of great interest since side chains often play an important role in protein folding and intermolecular interactions. A novel method for measuring the dynamics of methyl groups in uniformly ¹³C-, ¹⁵N-labeled proteins has been developed by our group. The method relies on the difference in peak intensities of ¹³C quartet components of methyl groups, in a spectrum recording the free evolution of ¹³C under proton coupling in a constant-time period. Cross-correlated relaxation rates between ¹³C-¹H dipoles can be easily measured from the intensities of the multiplet components. The degree of the methyl restrictions (S ²) can be estimated from the cross-correlated relaxation rate. The method is demonstrated on a sample of human fatty acid binding protein in the absence of fatty acid. We obtained relaxation data for 33 out of 46 residues having methyl groups in apo-IFABP. It has been found that the magnitude of the CSA tensor of spin ¹³C in a methyl group could be estimated from the intensities of the ¹³C multiplet components.     

1H, 15N and 13C resonance assignments,secondary structure,and the conformation of substrate in the binary folate complex of Escherichia coli dihydrofolate reductase

Summary By using fully ¹⁵N- and ¹⁵N/¹³C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific ¹H and ¹⁵N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the ¹³C resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected ¹⁵N and ¹³C experiments. A smaller but significant subset of side-chain ¹H and ¹³C assignments were also determined. In this complex, only one ¹⁵N or ¹³C resonance was detected per ¹⁵N or ¹³C protein nucleus, which indicated a single conformation. Proton-detected ¹³C experiments were also performed with unlabeled DHFR, complexed with ¹³C-7/¹³C-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on ¹H NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667–9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.     

1H, 13C, 15N resonance assignment of the chitin-binding protein CBP21 from Serratia marcescens

The 18.8 kDa chitin-binding protein CBP21 from Serratia marcescens has been isotopically labeled and recombinantly expressed. In this paper, we report the ¹H, ¹³C, ¹⁵N resonance assignment of CBP21.     

Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern

Oil palm has now become one of the most important crops, palm oil representing nearly 25% of global plant oil consumption. Many studies have thus addressed oil palm ecophysiology and photosynthesis‐based models of carbon allocation have been used. However, there is a lack of experimental data on carbon fixation and redistribution within palm trees, and important C‐sinks have not been fully characterized yet. Here, we carried out extensive measurement of natural ¹³C‐abundance (δ¹³C) in oil palm tissues, including fruits at different maturation stages. We find a ¹³C‐enrichment in heterotrophic organs compared to mature leaves, with roots being the most ¹³C‐enriched. The δ¹³C in fruits decreased during maturation, reflecting the accumulation in ¹³C‐depleted lipids. We further used observed δ¹³C values to compute plausible carbon fluxes using a steady‐state model of ¹³C‐distribution including metabolic isotope effects (¹²v/¹³v). The results suggest that fruits represent a major respiratory loss (≈39% of total tree respiration) and that sink organs such as fruits are fed by sucrose from leaves. That is, glucose appears to be a quantitatively important compound in palm tissues, but computations indicate that it is involved in dynamic starch metabolism rather that C‐exchange between organs.     

Sequence-specific 1H, 15N and 13C resonance assignments of Art v 1: a proline-rich allergen of Artemisia vulgaris pollen

Art v 1 is the major allergen of Artemisia vulgaris. The IgE raised against Art v 1 not only can cross-react with other proteins from the Asteraceae family members but also with components of various forms of food. Art v 1 is an important target for immunotherapy strategies, including vaccination with hypoallergenic derivatives or chimeras. We report the ¹H, ¹³C, and ¹⁵N resonance assignments of the recombinant Art v 1 and identification of secondary structures based on ¹³C chemical shifts.     

Synthesis of 4-thia-[6-13C]lysine from [2-13C]glycine: access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine

4-Thialysine (S-(2-aminoethyl)-l-cysteine) is an analog of lysine. It has been used as an alternative substrate for lysine in enzymatic reactions. Site-directed isotopomers are often needed for elucidation of mechanism of reactions. 4-Thialysine can be synthesized by reacting cysteine with 2-bromoethylamine, an important reagent in chemical-modification rescue (CMR) of proteins. Here, we present the synthesis of 4-thia-[6-¹³C]lysine, one of the isotopomers of 4-thialysine, from commercially available starting material [2-¹³C]glycine via formation of five intermediates including 2-amino[2-¹³C]ethanol and 2-bromo[1-¹³C]ethylamine. The compounds were characterized using various spectroscopic techniques. Moreover, we discuss that our strategy would provide access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine. Biological activity of 4-thia-[6-¹³C]lysine was tested in the enzymatic reaction of lysine 5,6-aminomutase.     

Disentangling CO2 fluxes: direct measurements of mesocosm‐scale natural abundance 13CO2/12CO2 gas exchange, 13C discrimination,and labelling of CO2 exchange flux components in controlled environments

A ¹³C/¹²C mass spectrometer was interfaced with a open gas exchange system including four growth chambers to investigate CO₂ exchange components of perennial ryegrass (Lolium perenne L.) stands. Chambers were fed with air containing CO₂ with known δ¹³C (δ_CΟ2?2.6 or ?46.8‰). The system did not fractionate C isotopes and no extraneous CO₂ leaked into chambers. The on‐line ¹³C discrimination (Δ) of ryegrass stands in light was independent of δ_CΟ2 when δ_CΟ2 was constant. The δ of CO₂ exchanged by the stands in light (δ_Nd) and darkness (δ_Rn) differed by 0.7‰, suggesting some Δ in dark respiration at the stand‐level. However, Δ decreased by ～ 10‰ when δ_CΟ2 was switched from ?46.8 to ?2.5‰, and increased by ～ 10‰ following a shift from ?2.6 to ?46.7‰ due to isotopic disequilibria between photosynthetic and respiratory fluxes. Isotopic imbalances were used to assess (non‐photorespiratory) respiration in light and the replacement of the respiratory substrate pool(s) by new photosynthate. Respiration was partially inhibited by light, but increased during the light period and decreased in darkness, in association with temperature changes. The labelling kinetics of respiratory CO₂ indicated the existence of two major respiratory substrate pools: a fast pool which was exchanged within hours, and a slow pool accounting for ～ 60% of total respiration and having a mean residence time of 3.6 d.     

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the ¹³C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-¹³C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the ¹³C enrichment. The approach to correct for these effects in metabolic flux studies using δ¹³C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and ¹³C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of ¹³C in proteinogenic amino acids over the whole range of applied concentrations of [1-¹³C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in ¹³C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.     

1H, 15N and 13C NMR assignments of mouse methionine sulfoxide reductase B2

A recombinant mouse methionine-r-sulfoxide reductase 2 (MsrB2ΔS) isotopically labeled with ¹⁵N and ¹⁵N/¹³C was generated. We report here the ¹H, ¹⁵N, and ¹³C NMR assignments of the reduced form of this protein. An erratum to this article can be found at