Quantitative isotopic C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: The case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin

Isotope fractionation is a powerful technique by which to probe the reaction mechanism of enzymes. The effect of a heavy isotope on the reaction energetics can be used to predict transition state architecture and reaction mechanism. In order to examine simultaneously the isotope fractionation in ¹³C at multiple sites within the substrate and product molecules without any need for site-selective isotope enrichment, a technique exploiting quantitative isotopic nuclear magnetic resonance (NMR) spectrometry at natural abundance (NAQ–NMR) has been developed. Here we report the first application of this technique to the study of an enzyme-catalyzed reaction, the bioconversion of ferulic acid to vanillin in cultures of Streptomyces setonii. We were able to show that the NAQ–NMR methodology is sufficiently precise and robust to measure the isotope shifts in the ¹³C/¹²C ratios in both substrate and product of this biotransformation, thereby permitting meaningful data to be obtained even at carbon positions that take part only indirectly in the reaction and show only secondary isotope fractionation. The results obtained provide direct evidence in support of the current hypothesis for the reaction mechanism of the enzyme hydroxycinnamoyl–CoA hydratase/lyase, notably the proposed involvement of the quinone methide enolate of feruloyl–CoA as intermediate in the catalytic pathway.     

The prediction of isotopic patterns in phenylpropanoids from their precursors and the mechanism of the NIH-shift: basis of the isotopic characteristics of natural aromatic compounds

The theoretical 2H-distribution in the aromatic ring of phenylpropanoids can be predicted from that of their precursors--erythrose-4-phosphate, phosphoenolpyruvate and NADPH--and by invoking the mechanism of the NIH-shift and implied deuterium isotope effects. For each position in the non-oxygenated ring, the predicted natural 2H-abundance is in excellent agreement with experimental data obtained from quantitative 2H NMR-measurements on natural compounds, especially concerning the relative 2H-abundances p > o > or = m. For the p-hydroxylated derivatives, the experimentally determined 2H-abundance sequence order m > o can also be deduced, assuming an anisotropic migration (intramolecular isotope effect) of the p-hydrogen atom to the two differently 2H-substituted m-positions during the NIH-shift (intramolecular hydrogen transfer) and an in vivo deuterium kinetic isotope effect of approximately 1.20 on the final hydrogen elimination from the proposed ketodiene intermediate. The predicted 2H-distribution pattern of methyl salicylate 10, a representative of an o-hydroxylated natural compound, is in excellent agreement with that reported from 2H NMR analyses. However, for salicyl alcohol, minor differences between the theoretical and experimentally determined pattern are found that cannot yet be satisfactorily explained. On the other hand, a very good agreement is found between the theoretical and experimental pattern of coumarin, provided a deuterium kinetic isotope effect of approximately 1.30 is assumed for the elimination of the H-atoms from the ketodiene intermediate. The secondary m-hydroxylation of p-coumaric acid in the biosynthesis of vanillin seems to proceed without large isotope effects. Parallel differences are also observed for the 18O-kinetic isotope effects on the corresponding monooxygenase-catalysed reactions. The results demonstrate convincingly that the mechanisms of these general reactions of the phenylpropanoid biosynthetic pathway are identical and follow general principles. Small observed differences between the 2H-patterns of individual natural aromatic compounds originating from the same hydroxylation type can therefore be assigned to differences of the patterns of the precursors, the extent and the orientation of the hydrogen migration, and the kinetic isotope effect on the final hydrogen elimination. The evidence for the existence of general systematic rules governing isotopic patterns in the shikimic acid pathway and its subsequent reactions is further supported by the recently reported 13C-distribution pattern of vanillin, which is also in agreement with that predicted from the precursors. Hence, it is apparent that the systematics of the isotope patterns of phenylpropanoids are in line with the generally accepted biosynthetic reactions in the shikimic acid pathway and that this knowledge can strengthen their value as an essential support for the distinction of natural and synthetic aromatic compounds.     

Natural Abundance Isotopic Fractionation in the Fermentation Reaction: Influence of the Nature of the Yeast

Site-specific natural isotope fractionation studied by nuclear magnetic resonance (SNIF-NMR) provides isotopic criteria that characterize a biochemical transformation such as fermentation and enable isotopic ratios measured in end products to be correlated with those of their precursors. In principle, a given set of transfer coefficients applies only to bioconversions performed under strictly identical conditions, a situation that is hardly fulfilled in most usual fermentation processes. In particular, natural raw materials such as fruits frequently involve complex mixtures of various yeast strains present at different concentrations. Series of experiments performed with different yeasts, different concentrations of car- bohydrates, and different yields of the transformation have shown that, although glycolysis is associated with overall hydrogen fractionation effects that may exceed 40 ppm, the range of variation in the isotopic ratios of the fermentation products, ethanol and water, does not exceed a few parts per million. Provided that the yield in ethanol reaches values higher than 70%, the nature of the yeast strain has minimal influence on the isotopic ratio of the methyl site of ethanol (D/H)_I. In contrast, the isotope ratio of the methylene site, (D/H)_II, may exhibit significant enhancements, in particular when ethanol is left in contact for a long time with poorly alcohologenic yeasts. These behaviors are consistent with hydrogen transfers from the aqueous medium to the methylene site, and partly to the methyl site, occurring with relatively high kinetic isotope effects. Since water acts as an open pool of hydrogens, however, only small isotopic variations are produced in the course of the fermentation reaction. Moreover, the partial connection between hydrogens from the methyl site of ethanol and hydrogens from glucose operates with relatively small secondary isotopic effects. No significant changes in the percentages of intra- and inter-molecular transfers of hydrogen to the methyl site are observed as a function of the nature of the yeast. These results support the use of the methyl isotopic ratio of ethanol as a probe of the isotopic behavior of carbohydrate precursors, whatever the yeast strains present in natural fermentation media.     

Measurement of 2H distribution in natural products by quantitative 2H NMR: An approach to understanding metabolism and enzyme mechanism?

During the biosynthesis of natural products, the intra-molecular distribution of isotopes is introduced as a result of different isotope effects associated with the reactions involved. Due to the sensitivity of certain enzymes to the presence of a heavy isotope, the isotope selection effects related to some transformations can be high, especially for hydrogen. The effect of a series of isotope effects specific to each enzyme-catalysed step are additive during a biosynthetic pathway, leading to fractionation of the isotopes between the starting substrate and the final product. As the individual reactions are acting on different positions in the substrate, the net effect is a non-statistical distribution of isotope within the final product. Quantitative ²H NMR spectroscopy can be used to measure the distribution of ²H at natural abundance in natural products. In the first example, the fermentation of glucose is examined. Glucose can act as a primary carbon source for a wide range of fermentation products, produced by a variety of pathways. In many cases, competing pathways are active simultaneously. The relative fluxes are influenced by both environmental and genetic parameters. Quantitative ²H NMR spectroscopy is being used to obtain mechanistic and regulatory information about isotopic fractionation from glucose during such fermentations. Quantitative ²H NMR spectroscopy can also be used to examine the fractionation in ²H that occurs in long-chain fatty acids during chain elongation and oxygenation. It has been found that the (²H/¹H) ratio shows an alternating pattern along the length of the chain and that the residual hydrogen atoms at the sites of desaturation are asymmetrically impoverished. The extent to which the non-statistical distribution of isotopes can be related to the mechanism of enzymes involved in the biosynthetic pathway via kinetic isotopic effects will be discussed.     

2H-NMR resolution of the methylenic isotopomers of ethanol applied to the study of stereospecific enzyme-catalysed exchange

We have shown that site-specific natural isotope fractionation of hydrogen studied by NMR (SNIF-NMR) is an important source of information on the mechanistic and environmental effects which govern the photosynthesis of sugars and their fermentation into ethanol. Three isotope ratios associated with the methyl, methylene, and hydroxyl sites of ethanol are determined in achiral media. In this study we show that complementary information about possible stereospecific mechanisms involving the methylenic hydrogens is also rendered accessible by 2H-NMR enantiomeric resolution. The synthesis of mandelate esters enables exchange between the pro-R site of ethanol and water to be investigated. Simultaneous access to the three site-specific isotope ratios of the ethyl group is obtained at isotopic dilutions close to the natural ones. Mediation of the exchange by the enzymic system alcohol dehydrogenase-alpha-lipoyldehydrogenase and by the yeast Saccharomyces cerevisiae are compared. The progress of the reaction can be followed quantitatively as a function of time and the occurrence of glycolytic metabolism of endogeneous materials by yeast can be substantiated in a one-pot experiment.     

Site-specific natural isotope fractionation of hydrogen in plant products studied by nuclear magnetic resonance

Deuterium NMR at the natural abundance was used to determine the site-specific isotope ratios (D/H)_i of the non-equivalent isotopomers of various chemical species which exist in plant products. The deuterium distribution in glucose, galactose and mannitol samples from different botanical and compartmental origins is discussed in terms of the influence of plant metabolism and environmental factors. Particular emphasis is given to the potential versatility of deuterium NMR in the study of natural isotopic distribution in pro-chiral situations. Typical examples of chiral recognition are given in the field of glycolysis metabolites (ethanol, amino-acids) and of monoterpene biosynthesis.     

A simplified GIS approach to modeling global leaf water isoscapes

The stable hydrogen (delta(2)H) and oxygen (delta(18)O) isotope ratios of organic and inorganic materials record biological and physical processes through the effects of substrate isotopic composition and fractionations that occur as reactions proceed. At large scales, these processes can exhibit spatial predictability because of the effects of coherent climatic patterns over the Earth's surface. Attempts to model spatial variation in the stable isotope ratios of water have been made for decades. Leaf water has a particular importance for some applications, including plant organic materials that record spatial and temporal climate variability and that may be a source of food for migrating animals. It is also an important source of the variability in the isotopic composition of atmospheric gases. Although efforts to model global-scale leaf water isotope ratio spatial variation have been made (especially of delta(18)O), significant uncertainty remains in models and their execution across spatial domains. We introduce here a Geographic Information System (GIS) approach to the generation of global, spatially-explicit isotope landscapes (= isoscapes) of "climate normal" leaf water isotope ratios. We evaluate the approach and the resulting products by comparison with simulation model outputs and point measurements, where obtainable, over the Earth's surface. The isoscapes were generated using biophysical models of isotope fractionation and spatially continuous precipitation isotope and climate layers as input model drivers. Leaf water delta(18)O isoscapes produced here generally agreed with latitudinal averages from GCM/biophysical model products, as well as mean values from point measurements. These results show global-scale spatial coherence in leaf water isotope ratios, similar to that observed for precipitation and validate the GIS approach to modeling leaf water isotopes. These results demonstrate that relatively simple models of leaf water enrichment combined with spatially continuous precipitation isotope ratio and climate data layers yield accurate global leaf water estimates applicable to important questions in ecology and atmospheric science.     

Kinetic isotope effects reveal the presence of significant secondary structure in the transition state for the folding of the N-terminal domain of L9

Our present understanding of the nature of the transition state for protein folding depends predominantly on studies where individual side-chain contributions are mapped out by mutational analysis (phi value analysis). This approach, although extremely powerful, does not in general provide direct information about the formation of backbone hydrogen bonds. Here, we report the results of amide H/D isotope effect studies that probe the development of hydrogen bonded interactions in the transition state for the folding of a small alpha-beta protein, the N-terminal domain of L9. Replacement of amide protons by deuterons in a solvent of constant isotopic composition destabilized the domain, decreasing both its T(m) and Delta G(0) of unfolding. The folding rate also decreased. The parameter Phi(H/D), defined as the ratio of the effect of isotopic substitution upon the activation free energy to the equilibrium free energy was determined to be 0.6 in a D(2)O background and 0.75 in a H(2)O background, indicating that significant intraprotein hydrogen bond interactions are developed in the transition state for the folding of NTL9. The value is in remarkably good agreement with more traditional measures of the position of the transition state, which report on the relative burial of surface area. The results provide a picture of a compact folding transition state containing significant secondary structure. Indirect analysis argues that the bulk of the kinetic isotope effect arises from the beta-sheet-rich region of the protein, and suggests that the development of intraprotein hydrogen bonds in this region plays a critical role in the folding of NTL9.     

Evaluation of isotope discrimination in C-based metabolic flux analysis

In a ¹³C experiment for metabolic flux analysis (¹³C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ¹³C) were in the range of −35 to −92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of ¹³C MFA.     

Isotope ratios of cellulose from plants having different photosynthetic pathways

Hydrogen and carbon isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from C₃, C₄, and Crassulacean acid metabolism (CAM) plants were determined for plants growing within a small area in Val Verde County, Texas. Plants having CAM had distinctly higher deuterium/hydrogen (D/H) ratios than plants having C₃ and C₄ metabolism. When hydrogen isotope ratios are plotted against carbon isotope ratios, each photosynthetic mode separates into a distinct cluster of points. C₄ plants had many D/H ratios similar to those of C₃ plants, so that hydrogen isotope ratios cannot be used to distinguish between these two photosynthetic modes. Portulaca mundula, which may have a modified photosynthetic mode between C₄ and CAM, had a hydrogen isotope ratio between those of the C₄ and CAM plants. When oxygen isotope ratios are plotted against carbon isotope ratios, no distinct clustering of the C₄ and CAM plants occurs. Thus, oxygen isotope ratios are not useful in distinguishing between these metabolic modes. A plot of hydrogen isotope ratios versus oxygen isotope ratios for this sample set shows considerable overlap between oxygen isotope ratios of the different photosynthetic modes without a concomitant overlap in the hydrogen isotope ratios of CAM and the other two photosynthetic modes. This observation is consistent with the hypothesis that higher D/H ratios in CAM plants relative to C₃ and C₄ plants are due to isotopic fractionations occurring during biochemical reactions.     

Carnosine as molecular probe for sensitive detection of Cu(II) ions using localized 1H NMR spectroscopy

Complex formation of carnosine (Csn) with Cu(II) is suspected to be of significant biochemical importance and can be detected by NMR via ion-induced paramagnetic relaxation of Csn signals. Here, we present quantification of the sensitivity achieved with localized (1)H NMR spectroscopy at physiological pH and high ligand-to-metal ratios. While characterizing the highly effective relaxation transfer onto a huge Csn pool due to fast ligand exchange, it is demonstrated that a metal-to-ligand ratio of approximately 100 ppm suffices to reduce Csn signals by approximately 50% in vitro, thus making the dipeptide a sensitive probe for such ions. Variation of the donor accessibility reveals that the paramagnetic effect is transferred onto a approximately 1370-fold donor abundance for a given ion concentration. A method is presented to characterize such effective ligand exchange relaxation transfer. These studies focus on the monomer formation since comparison with (1)H NMR data of human calf muscle demonstrates that the dimer complex is insignificant in vivo. Observed line broadening in living tissue yields an upper limit of ca. 195 ppm for the Csn-related copper concentration in human skeletal muscle.     

Conformational studies of a novel cationic glycolipid,glyceroplasmalopsychosine, from bovine brain by NMR spectroscopy

A novel glycosphingolipid containing a long chain aldehyde conjugated to galactose and glycerol, Gro1(3)-O-CH((CH(2))(n)CH(3))-O-6Galbeta-sphingosine (glyceroplasmalopsychosine) has been studied by NMR spectroscopy (Hikita et al. J. Biol. Chem. 2001, 276, 23084-23091). We further report here on the conformation showing the galactose and the glycerol at the end of two parallel hydrophobic chains, i.e. the sphingosine and the fatty aldehyde. This is proposed based on the interproton distances derived from ROESY experiments and 3 J (H,H) coupling constants. The absence of any intraresidual NOEs between protons in the glycerol residue suggested that the C-C-2 and C-C-3 bonds in the glycerol may be rotating freely, supporting the proposed conformation in which the unique terminal glycerol is in an environment with a minimal steric hindrance. The present study proposes a conformation of glyceroplasmalopsychosine greatly different from the two conventional plasmalopsychosines possessing a fatty aldehyde chain oriented in an opposite direction to the sphingosine.     

Comparison of measured and modeled variations in piñon pine leaf water isotopic enrichment across a summer moisture gradient

Stable hydrogen and oxygen isotopic composition of bulk leaf water (δD_lw and δ¹⁸O_lw) in piñon pine (Pinus edulis and P. monophylla) and gas exchange parameters were measured under field conditions to examine the effects of seasonal moisture stress on leaf water isotopic enrichment. Study sites were located near the lower elevation limit for piñon in the southwestern USA. Leaf-level transpiration measurements were made four times daily in spring, summer and early autumn; simultaneously, leaf samples were collected for water extraction and stable isotope analysis. Diurnal variations in δD_lw and δ¹⁸O_lw values were small, especially when leaf water residence times (molar leaf water content divided by transpiration rate) were high. Stomatal conductance explained most of the variance (60%) in leaf water enrichment across the dataset. Observed leaf water enrichment was compared with predictions of steady-state and nonsteady-state models. Nonsteady-state predictions fit observations the best, although D enrichment was often lower than predicted by any model. Hydrogen isotope ratios of leaf water and cellulose nitrate were strongly correlated, demonstrating preservation of a leaf water signal in wood and leaf cellulose.     

Hydrogen transfer pathways of the asymmetric reduction of alpha,beta-unsaturated ketone mediated by baker's yeast

The hydrogen transfer mechanism of cofactor reduction and recycling processes in the yeast reduction of alpha,beta-unsaturated ketone was studied by using quantitative isotope tracing close to natural abundance measured by (2)H NMR. In the reaction, the active cofactor is NADPH. The cofactor-transferred hydride attacks the beta sp(2) carbon of the enone carbonyl while water hydrogen is transferred to the alpha position. The reductant involved in the reaction depends on the quantity of yeast. When the amount of yeast is very large, the enzymes use preferentially certain unidentified substance stored in the yeast cells rather than the added glucose as electron donor. In this case, the hydrogen transferred by the cofactor is mainly of water origin. When the yeast amount is low, the added glucose is more efficiently used by the enzymes as electron donor and its hydrogen atoms bound to C-1 and C-3 are delivered to the substrate.     

Climatic significance of isotope ratios

The hydrogen and oxygen isotope ratios of water, which can be measured by Isotope Ratio Mass Spectrometry (IRMS), exhibit climatic dependencies and are commonly exploited in hydrogeology. More generally, the overall carbon or hydrogen isotope ratios of plant organic matter, and in particular of tree-ring cellulose, have been frequently used for climatic reconstruction. However, since many physicochemical and biochemical fractionation phenomena are likely to contribute to the isotopic values, the interpretation of the climatic significance of isotopic parameters is not always straightforward. In the case of hydrogen and oxygen for instance, the climatic profile of the source meteoric water is not simply transferred to leaf water and many steps of the biosyntheses are accompanied by kinetic and thermodynamic isotope effects that depend on the individual mechanistic pathways. The information brought about by overall isotope ratios determined by IRMS is averaged over all fractionation effects undergone at the different molecular positions. In contrast, the NMR investigation of Site-specific Natural Isotope Fractionation (SNIF-NMR) gives simultaneous access to isotope ratios specific to individual positions in the molecule. Since the different atoms do not necessarily exhibit the same climatic dependency, the method provides complementary responses to the environmental conditions. In particular, the isotopic parameters of ethanol and water obtained by fermenting sugars in standardized conditions reflect climatic influences which took place at different periods of plant growth. As a consequence, statistical analyses based on multi-site isotopic variables provide powerful criteria for distinguishing geographical regions of cultivation characterized by different climatic features. Although the sensitivity to climatic variations is the most pronounced for plant water and for sugars formed at the first step of photosynthesis, other components such as lipids or minor metabolites also exhibit climatic dependencies. The combination of isotopic values pertaining to different atomic species and either averaged over the whole molecule (IRMS) or associated with different molecular sites (SNIF-NMR), provides complementary criteria, which can be exploited in terms of both climatic significance and mechanistic pathways of the individual atoms.     

Conformational dynamics and solvent viscosity effects in carboxypeptidase-A-catalyzed benzoylglycylphenyllactate hydrolysis

We have used a new approach to the dynamics of hydrolytic metalloenzyme catalysis based on investigations of both external solvent viscosity effects and kinetic 2H isotope effects. The former reflects solvent and protein dynamics, and the nuclear reorganization distribution among damped protein motion and intramolecular friction-free nuclear motion. The isotope effect represents proton tunnelling and reorganization in the hydrogen bond network around the active site. We illustrate the approach by new spectrophotometric and pH-titration data for carboxypeptidase-A-catalyzed benzoylglycyl-L-phenyllactate hydrolysis. This substrate exhibits both a significant inverse fractional power law viscosity dependence over wide ranges controlled by glycerol and sucrose, and a kinetic 2H isotope effect of 1.65. The analogous benzoylglycylphenylalanine hydrolysis has a smaller isotope effect (1.3) and no viscosity dependence. Viscosity variation has no effect on the CD spectra in the 180-240-nm range. In terms of stochastic chemical rate theory, the data correspond to an enzyme-peptide substrate complex with a 'tight' structure protected from the solvent. In comparison, the enzyme-ester substrate complex is 'softer', strongly coupled to the solvent, and the rate-determining step is accompanied by proton transfer or by substantial reorganization in the hydrogen bonds near the active site.     

Metabolic processes account for the majority of the intracellular water in log-phase Escherichia coli cells as revealed by hydrogen isotopes

It is generally believed that water transport across biological membranes is essentially a near-instantaneous process, with water molecules diffusing directly across the membrane as well as through pores such as aquaporins. As a result of these processes by which water can equilibrate across a membrane, a common assumption is that intracellular water is isotopically indistinguishable from extracellular water. To test this assumption directly, we measured the hydrogen isotope ratio of intracellular water in Escherichia coli cells. Our results demonstrate that more than 50% of the intracellular water hydrogen atoms in log-phase E. coli cells are isotopically distinct from the growth medium water and that these isotopically distinct hydrogen atoms are derived from metabolic processes. As expected, the (2)H/(1)H isotope ratio of intracellular water from log-phase cells showed an appreciably larger contribution from metabolic water than did intracellular water from stationary-phase cells (53 +/- 12 and 23 +/- 5%, respectively). The (2)H/(1)H isotope ratio of intracellular water was also monitored indirectly by measuring the isotope ratio of fatty acids, metabolites that are known to incorporate hydrogen atoms from water during biosynthesis. Significantly, the difference in the isotopic composition of intracellular water from log- to stationary-phase E. coli cells was reflected in the hydrogen isotope ratio of individual fatty acids harvested at the two different times, indicating that the isotope ratio of metabolites can be used as an indirect probe of metabolic activity. Together, these results demonstrate that contrary to the common assumption that intracellular water is isotopically identical to extracellular water, these two pools of water can actually be quite distinct.     

Flight capabilities and feeding habits of silphine beetles: are flightless species really “carrion beetles”?

We determined the flight capabilities and feeding habits of adults of nine silphine beetle species and illustrated their relationship. We examined the silphine beetles for the presence or absence of flight muscles and estimated their feeding habits by comparing the carbon and nitrogen stable isotope ratios for them with those of necrophagous nicrophorine species and carnivorous carabine species. Three species (Silpha longicornis, S. perforata and Phosphuga atrata) completely lacked individuals with flight muscles, and one species (Eusilpha japonica) showed flight muscle dimorphism. Stable isotope analysis suggested that these species were carnivores, mainly feeding on soil invertebrates. Most flight species showed higher isotopic ratios than the flightless species. Some of them have isotopic ratios close to those of the nicrophorine species, suggesting that these species mainly feed on vertebrate carcasses. Flightless silphine species would have limited ability to search for patchy and unpredictable carcass resources. Further studies are necessary to understand the adaptive evolution of flight capability and the feeding habits in this group.     

Rooting depth,water availability,and vegetation cover along an aridity gradient in Patagonia

Above-and belowground biomass distribution, isotopic composition of soil and xylem water, and carbon isotope ratios were studied along an aridity gradient in Patagonia (44–45°S). Sites, ranging from those with Nothofagus forest with high annual rainfall (770 mm) to Nothofagus scrub (520 mm), Festuca (290 mm) and Stipa (160 mm) grasslands and into desert vegetation (125 mm), were chosen to test whether rooting depth compensates for low rainfall. Along this gradient, both mean above-and belowground biomass and leaf area index decreased, but average carbon isotope ratios of sun leaves remained constant (at-27), indicating no major differences in the ratio of assimilation to stomatal conductance at the time of leaf growth. The depth of the soil horizon that contained 90% of the root biomass was similar for forests and grasslands (about 0.80–0.50 m), but was shallower in the desert (0.30 m). In all habitats, roots reached water-saturated soils or ground water at 2–3 m depth. The depth profile of oxygen and hydrogen isotope ratios of soil water corresponded inversely to volumetric soil water contents and showed distinct patterns throughout the soil profile due to evaporation, water uptake and rainfall events of the past year. The isotope ratios of soil water indicated that high soil moisture at 2–3 m soil depth had originated from rainy periods earlier in the season or even from past rainy seasons. Hydrogen and oxygen isotope ratios of xylem water revealed that all plants used water from recent rain events in the topsoil and not from water-saturated soils at greater depth. However, this study cannot explain the vegetation zonation along the transect on the basis of water supply to the existing plant cover. Although water was accessible to roots in deeper soil layers in all habitats, as demonstrated by high soil moisture, earlier rain events were not fully utilized by the current plant cover during summer drought. The role of seedling establishment in determining species composition and vegetation type, and the indirect effect of seedling establishment on the use of water by fully developed plant cover, are discussed in relation to climate change and vegetation modelling.     

Automated extraction of backbone deuteration levels from amide H/2H mass spectrometry experiments

A Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high-resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side-chain hydrogens (present in MALDI-TOF H/2H data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side-chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances.