4-Hydroxybenzoate oligoprenyltransferase of E. coli, encoded in the gene ubiA, is an important key enzyme in the biosynthetic pathway to ubiquinone. It catalyzes the prenylation of 4-hydroxybenzoic acid in position 3 using an oligoprenyl diphosphate as a second substrate. Up to now, no X-ray structure of this oligoprenyltransferase or any structurally related enzyme is known. Knowledge of the tertiary structure and possible active sites is, however, essential for understanding the catalysis mechanism and the substrate specificity.With homology modeling techniques, secondary structure prediction tools, molecular dynamics simulations, and energy optimizations, a model with two putative active sites could be created and refined. One active site selected to be the most likely one for the docking of oligoprenyl diphosphate and 4-hydroxybenzoic acid is located near the N-terminus of the enzyme. It is widely accepted that residues forming an active site are usually evolutionary conserved within a family of enzymes. Multiple alignments of a multitude of related proteins clearly showed 100% conservation of the amino acid residues that form the first putative active site and therefore strongly support this hypothesis. However, an additional highly conserved region in the amino acid sequence of the ubiA enzyme could be detected, which also can be considered a putative (or rudimentary) active site. This site is characterized by a high sequence similarity to the aforementioned site and may give some hints regarding the evolutionary origin of the ubiA enzyme.Semiempirical quantum mechanical PM3 calculations have been performed to investigate the thermodynamics and kinetics of the catalysis mechanism. These results suggest a near SN1 mechanism for the cleavage of the diphosphate ion from the isoprenyl unit. The 4-hydroxybenzoic acid interestingly appears not to be activated as benzoate anion but rather as phenolate anion to allow attack of the isoprenyl cation to the phenolate, which appeared to be the rate limiting step of the whole process according to our quantum chemical calculations. Our models are a basis for developing inhibitors of this enzyme, which is crucial for bacterial aerobic metabolism.

Experimental and theoretical investigations concerning the second‐to‐last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants are reported. The proposed intrinsic or late intermediates 4‐oxo‐DMAPP ( 12 ) and 4‐hydroxy‐DMAPP ( 11 ) were synthesized in deuterium‐ or tritium‐labeled form according to new protocols especially adapted to work without protection of the diphosphate moiety. When the labeled compounds MEcPP ( 7 ), 11 , and 12 were applied to chromoplast cultures, aldehyde 12 was not incorporated. This finding is in agreement with a mechanistic and structural model of the responsible enzyme family: a three‐dimensional model of the fragment L271–A375 of the enzyme GcpE of Streptomyces coelicolor including NADPH, the Fe 4 S 4 cluster, and MEcPP ( 7 ) as ligand has been developed based on homology modeling techniques. The model has been accepted by the Protein Data Bank (entry code 1OX2). Supported by this model, semiempirical PM3 calculations were performed to analyze the likely catalysis mechanism of the reductive ring opening of MEcPP ( 7 ), hydroxyl abstraction, and formation of HMBPP ( 8 ). The mechanism is characterized by a proton transfer (presumably from a conserved arginine 286) to the substrate, accompanied by a ring opening without high energy barriers, followed by the transfer of two electrons delivered from the Fe 4 S 4 cluster, and finally proton transfer from a carboxylic acid side chain to the hydroxyl group to be removed from the ligand as water. The proposed mechanism is in agreement with all known experimental findings and the arrangement of the ligand within the enzyme. Thus, a very likely mechanism for the second to last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants is presented. A principally similar mechanism is also expected for the reductive dehydroxylation of HMBPP ( 8 ) to IPP ( 9 ) and DMAPP ( 10 ) in the last step.

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Changing environmental conditions, atmospheric pollutants and resistance reactions to pathogens cause production of reactive oxygen species (ROS) in plants. ROS in turn trigger the activation of signaling cascades such as the mitogen‐activated protein kinase (MAPK) cascade and accumulation of plant hormones, jasmonic acid, salicylic acid (SA), and ethylene (ET). We have used ozone (O3) to generate ROS in the apoplast of wild‐type Col‐0 and hormonal signaling mutants of Arabidopsis thaliana and show that this treatment caused a transient activation of 43 and 45 kDa MAPKs. These were identified as AtMPK3 and AtMPK6. We also demonstrate that initial AtMPK3 and AtMPK6 activation in response to O3 was not dependent on ET signaling, but that ET is likely to have secondary effects on AtMPK3 and AtMPK6 function, whereas functional SA signaling was needed for full‐level AtMPK3 activation by O3. In addition, we show that AtMPK3 , but not AtMPK6 , responded to O3 transcriptionally and translationally during O3 exposure. Finally, we show in planta that activated AtMPK3 and AtMPK6 are translocated to the nucleus during the early stages of O3 treatment. The use of O3 to induce apoplastic ROS formation offers a non‐invasive in planta system amenable to reverse genetics that can be used for the study of stress‐responsive MAPK signaling in plants.

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.