Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2,000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling.

A recently discovered, S‐adenosyl‐L ‐methionine and bivalent cation‐dependent O‐methyltransferase from the ice plant, Mesembryanthemum crystallinum , is involved in the methylation of various flavonoid and phenylpropanoid conjugates. Differences in regiospecificity as well as altered kinetic properties of the recombinant as compared to the native plant O‐methyltransferase can be attributed to differences in the N‐terminal part of the protein. Upon cleavage of the first 11 amino acids, the recombinant protein displays essentially the same substrate specificity as observed earlier for the native plant enzyme. Product formation of the newly designed, truncated recombinant enzyme is consistent with light‐induced accumulation of methylated flavonoid conjugates in the ice plant. Therefore, substrate affinity and regiospecificity of an O‐methyltransferase in vivo and in vitro can be controlled by cleavage of an N‐terminal domain.

Polymer-supported benzylhydrazines were synthesized using poly(ethylene glycol) acrylamide (PEGA) resin. They can be used to scavenge electrophiles reactive with hydrazine. Especially aromatic aldehydes can be captured selectively, monoprotected, and reversibly linked in the presence of other functional groups, including electrophilic ones. Various reactions can be performed on these protectively linked aldehydes, which afterward can be released either with full restoration of the aldehyde function or, alternatively, with simultaneous conversion.

4-Hydroxybenzoate oligoprenyl transferase from E. coli (ubiA-prenyl transferase) is a crucial enzyme for ubiquinone biosynthesis. It catalyzes the formation of 3-oligoprenyl-4-hydroxybenzoates like geranyl hydroxybenzoate (GHB, 23) from geranyl pyrophosphate (GPP, 22). Several analogues and mimics of geranyl pyrophosphate have been prepared for an examination of their ability to inhibit the enzyme. 7,11-Dimethyl-3-oxododeca-6,10-dienoic acid (2), 3-hydroxy7,11- dimethyldodeca-6,10-dienoic acid (3), 2-hydroxy-4,8-dimethyl-3,7-nonadienylphosphonic acid (4), and tripotassium [[(4E)-5,9-dimethyldeca-4,8-dienyl]phosphinato](difluoro)methylphosphonate (5) were synthesized from geraniol. .-2,.-1-Dihydroxylated farnesyl diphosphate 6 was prepared from trans,trans-farnesol. All compounds were tested for enzyme inhibition in a competitive assay with natural substrate. The effect of these compounds on ubiA-prenyltransferase activity varied substantially, ranging from almost full inhibition to, surprisingly, enhanced enzymatic activity at low concentrations by some compounds. A special, EDTAmodifyable magnesium effect is discussed as potential reason.

This chapter presents jasmonates and their related compounds and discusses jasmonate-induced alteration of gene expression. Jasmonates exerts two different changes in gene expression— decrease in the expression of nuclear- and chloroplast-encoded genes and increase in the expression of specific genes. Jasmonates are shown to alter sink-source relationships such as JA promotes formation of the N-rich vegetative storage proteins—VSPα and VSPβ—of soybean, including reallocation in pod filling. In addition to such nutrient reallocation to other parts of the plant, jasmonates cause decreases in photosynthesis and chlorophyll content, the most significant manifestations of senescence in leaves. The rise of endogenous jasmonates upon stress or exogenous treatment with jasmonates correlates in time with the expression of various genes. The promotion of senescence by jasmonates is counteracted by cytokinins. The capacity of jasmonates to down regulate photosynthetic genes may also be one determinant in the onset of senescence.

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Conformational analysis by NMR spectroscopy and molecular modeling revealed a left-handed PPII helix-like structure for Trp2-Tat(1–9) (cis and trans) and an even more flexible structure for TXA2-R(1–9).PPII helices form a well-defined structural class comparable with the other structures defined in proteins and are characterized by exposed, mobile structures with 4–8 residues, mostly found on the protein surface. Polyproline II helices are mainly identified by their torsion angles of φ∼−75° and Ψ∼145−. They do not form regular interchain hydrogen bonds, but are hydrogen bonded with water molecules. PPII helices have a strong preference for the amino acid proline, although it is not necessarily present. These features were also reported for the parent peptide Tat(1–9)4 as well as for the well known DP IV substrates neuropeptide Y and pancreatic polypeptide5 suggesting that PPII-like helical structures represent a favored structural class for the interaction with DP IV.Thus, the considerable enhancement of the inhibition capacity of both Trp2-Tat(1–9) and TXA2-R(1–9) compared to the moderate inhibitor Tat(1–9)2, Ki=2.68±0.01 10−4 M, can only be due to tryptophan in the second position suggesting that its side chain is favored to exhibit attractive hydrophobic interactions with DP IV compared with aspartic acid.On the other hand, we could show recently that Tat(1–9) and its analogues as well as TXA2-R(1–9) inhibit DP IV according to different inhibition mechanisms (Lorey et al., manuscript submitted). One possible explanation for these findings might be enzyme-ligand interactions relying on multiple weak binding sites as described for PPII helices5 rather than specific lock and key binding. Certainly, only an X-ray structure of DP IV would help to understand the interaction of DP IV with inhibitors.

Inhibition of the enzymatic activity of alanyl-aminopeptidase leads to strong immunosuppression both in vitro and in vivo. Mechanisms involved include growth arrest, induction of immunosuppressive cytokines (TGF-ß1), reduced expression of inflammatory or T cell stimulating cytokines (IL-2, IL-12), and modulation of T cell signalling pathways. Thus, T cells appear to represent a major cellular target for the pharmacological treatment of T cell mediated diseases by virtue of aminopeptidase inhibitor administration. Membrane (APN) and cytosol alanyl-aminopeptidase (ApPS), both implicated in a variety of cellular functions, show similar substrate specifity and inhibitor sensitivity. Furthermore, both enzymes are expressed in practically all T cell subsets, including the population of natural regulatory T cells that was shown recently to control the immunological tolerance to self-antigens. While the involvement of APN and ApPS in the pathological immune response is evident, the precise molecular mechanisms remain to be identified. The development of inhibitors specific for APN and ApPS is an attractive field of study and would allow determination of the individual contribution of either enzyme in the immune response.

Our results indicate that the substrate properties of peptides are encoded by their own structure. That means, that substrate characteristics depend not only on the primary structure around the catalytic site rather C-terminal located secondary interactions strongly influence the binding and catalysis of the substrates. Such interaction sites seem to force the ligand in a proper orientation to the active site of DP IV. As result of these relations the hydrolysis of peptides with non-proline and non-alanine residues in P1-position (Ser, Val, Gly) becomes possible in longer peptides.Such specific secondary interactions opens the opportunity for development of new inhibitors.