Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g’-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.

In the last decade thiotaurine, 2-aminoethane thiosulfonate, has been investigated as an inflammatory modulating agent as a result of its ability to release hydrogen sulfide (H2S) known to play regulatory roles in inflammation. Thiotaurine can be included in the “taurine family” due to structural similarity to taurine and hypotaurine, and is characterized by the presence of a sulfane sulfur moiety. Thiotaurine can be produced by different pathways, such as the spontaneous transsulfuration between thiocysteine – a persulfide analogue of cysteine – and hypotaurine as well as in vivo from cystine. Moreover, the enzymatic oxidation of cysteamine to hypotaurine and thiotaurine in the presence of inorganic sulfur can occur in animal tissues and last but not least thiotaurine can be generated by the transfer of sulfur from mercaptopyruvate to hypotaurine catalyzed by a sulfurtransferase. Thiotaurine is an effective antioxidant agent as demonstrated by its ability to counteract the damage caused by pro-oxidants in the rat. Recently, we observed the influence of thiotaurine on human neutrophils functional responses. In particular, thiotaurine has been found to prevent human neutrophil spontaneous apoptosis suggesting an alternative or additional role to its antioxidant activity. It is likely that the sulfane sulfur of thiotaurine may modulate neutrophil activation via persulfidation of target proteins. In conclusion, thiotaurine can represent a biologically relevant sulfur donor acting as a biological intermediate in the transport, storage and release of sulfide.

Conditional gene expression and modulating protein stability under physiological conditions are important tools in biomedical research. They led to a thorough understanding of the roles of many proteins in living organisms. Current protocols allow for manipulating levels of DNA, mRNA, and of functional proteins. Modulating concentrations of proteins of interest, their post-translational processing, and their targeted depletion or accumulation are based on a variety of underlying molecular modes of action. Several available tools allow a direct as well as rapid and reversible variation right on the spot, i.e., on the level of the active form of a gene product. The methods and protocols discussed here include inducible and tissue-specific promoter systems as well as portable degrons derived from instable donor sequences. These are either constitutively active or dormant so that they can be triggered by exogenous or developmental cues. Many of the described techniques here directly influencing the protein stability are established in yeast, cell culture and in vitro systems only, whereas the indirectly working promoter-based tools are also commonly used in higher eukaryotes. Our major goal is to link current concepts of conditionally modulating a protein of interest’s activity and/or abundance and approaches for generating cell and tissue types on demand in living, multicellular organisms with special emphasis on plants.

Multi-component reactions of building blocks with more than one MCR-reactive group will give rise to oligomeric MCR products. The proper choice of at least two bifunctional building blocks will give either a polymeric or a cyclic product. Apart from polymerization, repetitive or consecutive Ugi reactions have been used to produce linear MCR-heterooligomers with such building blocks.

Glucosinolates (GLSs) present in Brassica vegetables serve as precursors for biologically active metabolites, which are released by myrosinase and induce phase 2 enzymes via the activation of Nrf2. Thus, GLSs are generally considered beneficial. The pattern of GLSs in plants is various, and contents of individual GLSs change with growth phase and culture conditions. Whereas some GLSs, for example, glucoraphanin (GRA), the precursor of sulforaphane (SFN), are intensively studied, functions of others such as the indole GLS neoglucobrassicin (nGBS) are rather unknown as are functions of combinations thereof. We therefore investigated myrosinase-treated GRA, nGBS and synthetic SFN for their ability to induce NAD(P)H:quinone oxidoreductase 1 (NQO1) as typical phase 2 enzyme, and glutathione peroxidase 2 (GPx2) as novel Nrf2 target in HepG2 cells. Breakdown products of nGBS potently inhibit both GRA-mediated stimulation of NQO1 enzyme and Gpx2 promoter activity. Inhibition of promoter activity depends on the presence of an intact xenobiotic responsive element (XRE) and is also observed with benzo[a]pyrene, a typical ligand of the aryl hydrocarbon receptor (AhR), suggesting that suppressive effects of nGBS are mediated via AhR/XRE pathway. Thus, the AhR/XRE pathway can negatively interfere with the Nrf2/ARE pathway which has consequences for dietary recommendations and, therefore, needs further investigation.

0

Several mammalian peptide hormones and proteins from plant and animal origin contain an N-terminal pyroglutamic acid (pGlu) residue. Frequently, the moiety is important in exerting biological function in either mediating interaction with receptors or stabilizing against N-terminal degradation. Glutaminyl cyclases (QCs) were isolated from different plants and animals catalyzing pGlu formation. The recent resolution of the 3D structures of Carica papaya and human QCs clearly supports different evolutionary origins of the proteins, which is also reflected by different enzymatic mechanisms. The broad substrate specificity is revealed by the heterogeneity of physiological substrates of plant and animal QCs, including cytokines, matrix proteins and pathogenesis-related proteins. Moreover, recent evidence also suggests human QC as a catalyst of pGlu formation at the N-terminus of amyloid peptides, which contribute to Alzheimer's disease. Obviously, owing to its biophysical properties, the function of pGlu in plant and animal proteins is very similar in terms of stabilizing or mediating protein and peptide structure. It is possible that the requirement for catalysis of pGlu formation under physiological conditions may have triggered separate evolution of QCs in plants and animals.

In selenocysteine (Sec, U)-containing proteins the selenenylsulfide bridge and its reduced thiol-selenol counterpart are usually the significant species. An important role for serine as flanking amino acid in the redox potential of S-S and S-Se bridges was proposed for some thioredoxin reductases. To check the generality of this proposal, model tetrapeptides (GCCG, SCCG, GCCS, SCCS, GCUG, SCUG, GCUS, SCUS) were synthesized, including the GCUG sequence of human thioredoxin reductase. The influence on the redox potential of S-Se and S-S bridges as a function of pH and of serine at different positions reveals (i) a strong general pH dependence, and (ii) a significant influence of flanking serine on disulfide only at basic pH.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana. Upon expression of the corresponding cDNAs from both plants via the secretory pathway of Pichia pastoris, two active QC proteins were isolated. The specificity of the purified proteins was assessed using various substrates with different amino acid composition and length. Highest specificities were observed with substrates possessing large hydrophobic residues adjacent to the N-terminal glutamine and for fluorogenic dipeptide surrogates. However, compared to Carica papaya QC, the specificity constants were approximately one order of magnitude lower for most of the QC substrates analyzed. The QCs also catalyzed the conversion of N-terminal glutamic acid to pyroglutamic acid, but with approximately 105- to 106-fold lower specificity. The ubiquitous distribution of plant QCs prompted a search for potential substrates in plants. Based on database entries, numerous proteins, e.g., pathogenesis-related proteins, were found that carry a pyroglutamate residue at the N-terminus, suggesting QC involvement. The putative relevance of QCs and pyroglutamic acid for plant defense reactions is discussed.

What makes selenoenzymes – seen from a chemist's view – so special that they cannot be substituted by just more analogous or adapted sulfur proteins? This review compiles and compares physicochemical properties of selenium and sulfur, synthetic routes to selenocysteine (Sec) and its peptides, and comparative studies of relevant thiols and selenols and their (mixed) dichalcogens, required to understand the special role of selenium in selenoproteins on the atomic molecular level. The biochemically most relevant differences are the higher polarizability of Se- and the lower pKa of SeH. The latter has a strikingly different pH-dependence than thiols, with selenols being active at much lower pH. Finally, selected typical enzymatic mechanisms which involve selenocysteine are critically discussed, also in view of the authors' own results.