Bent O. Petersen

An NMR-based metabonomic investigation on effects of milk and meat protein diets given to 8-year-old boys

The objective of the study was to investigate the ability of an NMR-based metabonomic approach, applied to biofluids, to explore and identify overall exogenous and endogenous biochemical effects of a short-time high intake of milk protein or meat protein given to prepubertal children, the aim being to compare relative differences and not an absolute quantification. A total of twenty-four 8-year-old boys were asked to take 53 g protein as milk (n 12) or meat daily (n 12). At baseline and after 7 d, urine and serum samples were collected and high-resolution 1H NMR spectra were acquired on these using a 800 MHz spectrometer. The milk diet reduced the urinary excretion of hippurate, while the meat diet increased the urinary excretion of creatine, histidine and urea. The NMR measurements on serum revealed minor changes in the lipid profile, which most probably should be ascribed to an increase in the content of SCFA in the blood after consumption of the milk diet. The meat diet had no effect on the metabolic profile of serum. The study for the first time demonstrates the capability of proton NMR-based metabonomics to identify the overall biochemical effects of consumption of different animal proteins. The urine metabolite profile is more susceptible to perturbations as a result of short diet interventions than the serum metabolite profile. The milk diet-induced reduction in urinary excretion of hippurate suggests alterations in gut microflora, which may be useful information for further studies elucidating the effects of bioactive components in milk.

Metabolomic Signatures of Inbreeding at Benign and Stressful Temperatures in Drosophila melanogaster

While the population genetics of inbreeding is fairly well understood, the effects of inbreeding on the physiological and biochemical levels are not. Here we have investigated the effects of inbreeding on the Drosophila melanogaster metabolome. Metabolite fingerprints in males from five outbred and five inbred lines were studied by nuclear magnetic resonance spectroscopy after exposure to benign temperature, heat stress, or cold stress. In both the absence and the presence of temperature stress, metabolite levels were significantly different among inbred and outbred lines. The major effect of inbreeding was increased levels of maltose and decreased levels of 3-hydroxykynurenine and a galactoside [1-O-(4-O-(2-aminoethyl phosphate)-β-d-galactopyranosyl)-x-glycerol] synthesized exclusively in the paragonial glands of Drosophila species, including D. melanogaster. The metabolomic effect of inbreeding at the benign temperature was related to gene expression data from the same inbred and outbred lines. Both gene expression and metabolite data indicate that fundamental metabolic processes are changed or modified by inbreeding. Apart from affecting mean metabolite levels, inbreeding led to an increased between-line variation in metabolite profiles compared to outbred lines. In contrast to previous observations revealing interactions between inbreeding and environmental stress on gene expression patterns and life-history traits, the effect of inbreeding on the metabolite profile was similar across the different temperature treatments.

Complete Structures of Bordetella bronchiseptica and Bordetella parapertussis Lipopolysaccharides

The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry. This identified a novel pentasaccharide fragment that links the O chain to the core in all the LPS studied. In addition, although the O chain of these bacteria was reported as a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-α-galacturonic acid, we discovered that the polymer contains several amidated uronic acids, the number of which varies between strains. These new data describe the complete structure of the LPS carbohydrate backbone for both Bordetella species and help to explain the complex genetics of LPS biosynthesis in these bacteria.

Improved Characterization of Nod Factors and Genetically Based Variation in LysM Receptor Domains Identify Amino Acids Expendable for Nod Factor Recognition in Lotus spp.

Formation of functional nodules is a complex process depending on host-microsymbiont compatibility in all developmental stages. This report uses the contrasting symbiotic phenotypes of Lotus japonicus and L. pedunculatus, inoculated with Mesorhizobium loti or the Bradyrhizobium sp. (Lotus), to investigate the role of Nod factor structure and Nod factor receptors (NFR) for rhizobial recognition, infection thread progression, and bacterial persistence within nodule cells. A key contribution was the use of 800 MHz nuclear magnetic resonance spectroscopy and ultrahigh-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry for Nod factor analysis. The Nod factor decorations at the nonreducing end differ between Bradyrhizobium sp. (Lotus) and M. loti, and the NFR1/NFR5 extracellular regions of L. pedunculatus and L. japonicus were found to vary in amino acid composition. Genetic transformation experiments using chimeric and wild-type receptors showed that both receptor variants recognize the structurally different Nod factors but the later symbiotic phenotype remained unchanged. These results highlight the importance of additional checkpoints during nitrogen-fixing symbiosis and define several amino acids in the LysM domains as expendable for perception of the two differentially carbamoylated Nod factors.

The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel α‐glucosides through reverse phosphorolysis by maltose phosphorylase

A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin‐binding protein, three maltodextrin ATP‐binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI‐GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing β‐glucose 1‐phosphate (β‐Glc 1‐P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and β‐Glc 1‐P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3‐OH and 4‐OH, such as glucose and mannose, and also reacted with 2‐deoxy glucosamine and 2‐deoxy N‐acetyl glucosamine. By contrast, none of the tested di‐ and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mmβ‐Glc 1‐P and 50 mm glucose, glucosamine, N‐acetyl glucosamine, mannose, xylose or l‐fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI‐MS to be α‐Glcp‐(1→4)‐Glcp (maltose), α‐Glcp‐(1→4)‐GlcNp (maltosamine), α‐Glcp‐(1→4)‐GlcNAcp (N‐acetyl maltosamine), α‐Glcp‐(1→4)‐Manp, α‐Glcp‐(1→4)‐Xylp and α‐Glcp‐(1→4)‐ l‐Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

Characterization of the lipopolysaccharide and β‐glucan of the fish pathogen Francisella victoria

Lipopolysaccharide (LPS) and β‐glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods. The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3‐aminobutyric acid, and a novel branched amino acid, thus confirming F. victoria as a unique species. The structure identified composes the largest oligosaccharide elucidated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, including the first application of H2BC sequence to oligosaccharides. The non‐phosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found in Francisella novicida. The heptoseless core‐lipid A region of the LPS contained a linear pentasaccharide fragment identical to the corresponding part of F. tularensis and F. novicida LPSs, differing in side‐chain substituents. The linkage region of the O‐chain also closely resembled that of other Francisella. LPS preparation contained two characteristic glucans, previously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual β‐(1–6)‐glucan with (glycerol)2phosphate at the reducing end.

Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum.

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl](n)-(1→2)-D-glucopyranose, and β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl](n)-(1→3)-D-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.

Structural characterization of homogalacturonan by NMR spectroscopy-assignment of reference compounds.

Complete assignment of (1)H and (13)C NMR of six hexagalactopyranuronic acids with varying degree and pattern of methyl esterification is reported. The NMR experiments were run at room temperature using approximately 2mg of sample making this method convenient for studying the structure of homogalacturonan oligosaccharides.

Study of molecular interactions with 13C DNP-NMR

NMR spectroscopy is an established, versatile technique for the detection of molecular interactions, even when these interactions are weak. Signal enhancement by several orders of magnitude through dynamic nuclear polarization alleviates several practical limitations of NMR-based interaction studies. This enhanced non-equilibrium polarization contributes sensitivity for the detection of molecular interactions in a single NMR transient. We show that direct (13)C NMR ligand binding studies at natural isotopic abundance of (13)C gets feasible in this way. Resultant screens are easy to interpret and can be performed at (13)C concentrations below muM. In addition to such ligand-detected studies of molecular interaction, ligand binding can be assessed and quantified with enzymatic assays that employ hyperpolarized substrates at varying enzyme inhibitor concentrations. The physical labeling of nuclear spins by hyperpolarization thus provides the opportunity to devise fast novel in vitro experiments with low material requirement and without the need for synthetic modifications of target or ligands.

Nuclear magnetic resonance–based metabonomics reveals strong sex effect on plasma metabolism in 17-year–old Scandinavians and correlation to retrospective infant plasma parameters

Nuclear magnetic resonance (NMR)-based metabonomics was carried out on plasma samples from a total of seventy-five 17-year-old Danes to investigate the impact of key parameters such as sex, height, weight, and body mass index on the plasma metabolite profile in a normal, healthy population. Principal component analysis identified sex to have a large impact on the NMR plasma metabolome, whereas no apparent effects of height, weight, and body mass index were found. Partial least square regression discriminant analysis and quantification of relative metabolite concentrations by integration of NMR signals revealed that the sex effect included differences in plasma lipoproteins (mainly high-density lipoprotein), glucose, choline, and amino acid content. Accordingly, the present study suggests a higher lipid synthesis in young women than young men and a higher protein turnover in young men compared with women. Data on plasma content of triglyceride, lipoprotein fractions, and cholesterol at an age of 9 months were available for selected individuals (n = 40); and partial least square regressions revealed correlations between these infant parameters and the NMR plasma metabolome at an age of 17 years. In conclusion, the present study demonstrates the feasibility of NMR-based metabonomics for obtaining a deeper insight into interindividual differences in metabolism and for exploring relationships between parameters measured early in life and metabolic status at a later stage.