Substrate and inhibitor spectra of ethylbenzene dehydrogenase: perspectives on application potential and catalytic mechanism

Ethylbenzene dehydrogenase (EbDH) catalyzes the initial step in anaerobic degradation of ethylbenzene in denitrifying bacteria, namely, the oxygen-independent hydroxylation of ethylbenzene to (S)-1-phenylethanol. In our study we investigate the kinetic properties of 46 substrate analogs acting as substrates or inhibitors of the enzyme. The apparent kinetic parameters of these compounds give important insights into the function of the enzyme and are consistent with the predicted catalytic mechanism based on a quantum chemical calculation model. In particular, the existence of the proposed substrate-derived radical and carbocation intermediates is substantiated by the formation of alternative dehydrogenated and hydroxylated products from some substrates, which can be regarded as mechanistic models. In addition, these results also show the surprisingly high diversity of EbDH in hydroxylating different kinds of alkylaromatic and heterocyclic compounds to the respective alcohols. This may lead to attractive industrial applications of ethylbenzene dehydrogenase for a new process of producing alcohols via hydroxylation of the corresponding aromatic hydrocarbons rather than the customary procedure of reducing the corresponding ketones.     

Development of a bacterial system for high yield expression of fully functional adrenal cytochrome b561

Adrenal cytochrome b561 (cyt b561) is the prototypical member of an emerging family of proteins that are distributed widely in vertebrate, invertebrate and plant tissues. The adrenal cytochrome is an integral membrane protein with two b-type hemes and six predicted transmembrane helices. Adrenal cyt b561 is involved in catecholamine biosynthesis, shuttling reducing equivalents derived from ascorbate. We have developed an Escherichia coli system for expression, solubilization and purification of the adrenal cytochrome. The spectroscopic and redox properties of the purified recombinant protein expressed in this prokaryotic system confirm that the cytochrome retains a native, fully functional form over a wide pH range. Mass spectral analysis shows that the N-terminal signal peptide is intact. The new bacterial expression system for cyt b561 offers a sixfold improvement in yield and other substantial advantages over existing insect and yeast cell systems for producing the recombinant cytochrome for structure-function studies.     

Peptaibiomics: microheterogeneity, dynamics, and sequences of trichobrachins, peptaibiotics from Trichoderma parceramosum Bissett (T. longibrachiatum Rifai)

From the culture broth of the filamentous fungus Trichoderma parceramosum, strain CBS 936.69, a mixture of polypeptide antibiotics (pepaibiotics), named trichobrachin (TB), was isolated. Three major groups designated TB I, TB II, and TB III could be separated and isolated by preparative TLC on silica gel. Individual peptides of these three groups were sequenced by on-line LC/ESI-MS(n). The mixture of N-acetylated peptides comprises ten 19-residue peptides with a free C-terminal Gln residue (TB I peptides), two 18-residue peptides with a free C-terminal Gln residue (TB II 1 and 2), seven 20-residue peptides with a C-terminal amide-bound phenylalaninol (TB II 3-10), and 34 eleven-residue peptides with either a C-terminal leucinol or isoleucinol or valinol (TB III 1-34). Monitoring production and degradation of peptaibiotics in a pilot experiment revealed that the biosynthesis of TB II and TB III peptides starts two days after the beginning of fermentation. After five days of fermentation, the concentration of TB II decreased, whereas the amount of TB I increased. This observation unequivocally demonstrates that those two 18-residue TB I and TB II peptides with the free carboxy terminus result from enzymatic C-terminal degradation of the 20-residue TB II peptides. In analogy to the technical terms proteome and proteomics, the terms peptaibiome and peptaibiomics have recently been proposed for the entirety and dynamics of the Aib-containing peptides (comprehensively named peptaibiotics). Consequently, the entire peptaibiome of T. parceramosum grown under submerse conditions in shake-flasks for five days comprises at least 54 peptides differing in main-chain length and microheterogeneity, i.e., exchange of amino acids and the C-terminal 1,2-amino alcohol.     

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.     

Antiproliferative effect and genotoxicity of novel synthesized palladium complexes with organoarsenic ligands

Three new palladium complexes with general formula [PdCl₂L₂], where L = heterofunctional organoarsenic ligand: (2-isopropoxyphenyl)diphenylarsine (1), (2-methoxyphenyl)-diphenylarsine (2) and (2-hydroxyphenyl)diphenylarsine (3) have been synthesized and fully characterized, including X-ray crystallographic data. Their potential antitumor effect and genotoxicity have been studied as well. The viability test performed on human tumor (MLS) and normal (Hfl-1) cell lines indicates significant cytotoxicity of complexes, which is higher in tumor cells than in normal cells. The lethal doses are comparable with those of standard metal-based chemotherapeutical drugs (carboplatin and oxaliplatin). These palladium complexes exhibit a higher cytotoxicity against tumor cells as against normal cells in vitro. A new static cytometric method was developed and simultaneously the classic AnnexinV test was performed. Complex 2 has an important capacity to induce apoptosis in tumor cells. The apoptotic process is triggered due to the interaction of these complexes with secondary structure of DNA in treated cells. The alkaline single-cell gel assay shows that the level of DNA damages induced by compounds 2 and 3 are significantly higher in tumor cells as in normal cells. These studies shown that complexes 1, 2 and 3 have biologic activity, the effect of complex 2 being superior to its platinum analogues, attributable to its structure.     

Peroxide-induced radical formation at TYR385 and TYR504 in human PGHS-1

Prostaglandin H synthase isoforms 1 and -2 (PGHS-1 and -2) react with peroxide to form a radical on Tyr385 that initiates the cyclooxygenase catalysis. The tyrosyl radical EPR signals of PGHS-1 and -2 change over time and are altered by cyclooxygenase inhibitor binding. We characterized the tyrosyl radical dynamics using wild type human PGHS-1 (hPGHS-1) and its Y504F, Y385F, and Y385F/Y504F mutants to determine whether the radical EPR signal changes involve Tyr504 radical formation, Tyr385 radical phenyl ring rotation, or both. Reaction of hPGHS-1 with peroxide produced a wide singlet, whereas its Y504F mutant produced only a wide doublet signal, assigned to the Tyr385 radical. The cyclooxygenase specific activity and K_M value for arachidonate of hPGHS-1 were not affected by the Y504F mutation, but the peroxidase specific activity and the K_M value for peroxide were increased. The Y385F and Y385F/Y504F mutants retained only a small fraction of the peroxidase activity; the former had a much-reduced yield of peroxide-induced radical and the latter essentially none. After binding of indomethacin, a cyclooxygenase inhibitor, hPGHS-1 produced a narrow singlet but the Y504F mutant did not form a tyrosyl radical. These results indicate that peroxide-induced radicals form on Tyr385 and Tyr504 of hPGHS-1, with radical primarily on Tyr504 in the wild type protein; indomethacin binding prevented radical formation on Tyr385 but allowed radical formation on Tyr504. Thus, hPGHS-1 and -2 have different distributions of peroxide-derived radical between Tyr385 and Tyr504. Y504F mutants in both hPGHS-1 and -2 significantly decreased the cyclooxygenase activation efficiency, indicating that formation of the Tyr504 radical is functionally important for both isoforms.     

The structural basis for peptide selection by the transport receptor OppA

Oligopeptide‐binding protein A (OppA) from Lactococcus lactis binds peptides of an exceptionally wide range of lengths (4–35 residues), with no apparent sequence preference. Here, we present the crystal structures of OppA in the open‐ and closed‐liganded conformations. The structures directly explain the protein's phenomenal promiscuity. A huge cavity allows binding of very long peptides, and a lack of constraints for the position of the N and C termini of the ligand is compatible with binding of peptides with varying lengths. Unexpectedly, the peptide's amino‐acid composition (but not the exact sequence) appears to have a function in selection, with a preference for proline‐rich peptides containing at least one isoleucine. These properties can be related to the physiology of the organism: L. lactis is auxotrophic for branched chain amino acids and favours proline‐rich caseins as a source of amino acids. We propose a new mechanism for peptide selection based on amino‐acid composition rather than sequence.