The reversible oxidation of methionine residues in proteins has emerged as a biologically important post-translational modification. However, detection and quantitation of methionine sulfoxide in proteins is difficult. Our aim is to develop a method for specifically derivatizing methionine sulfoxide residues. We report a Pummerer rearrangement of methionine sulfoxide treated sequentially with trimethylsilyl chloride and then 2-mercaptoimidazole or pyridine-2-thiol to produce a dithioacetal product. This derivative is stable to standard mass spectrometry conditions, and its formation identified oxidized methionine residues. The scope and requirements of dithioacetal formation are reported for methionine sulfoxide and model substrates. The reaction intermediates have been investigated by computational techniques and by 13C NMR spectroscopy. These provide evidence for an α-chlorinated intermediate. The derivatization allows for detection and quantitation of methionine sulfoxide in proteins by mass spectrometry and potentially by immunochemical methods. 相似文献
The Rhodococcus jostii RHA1 genome encodes a number of enzymes that can be exploited as biocatalysts. Study of the substrate spectrum and enantioselectivity of Baeyer–Villiger monooxygenases from R. jostii allowed the identification of short amino acid sequences specific to groups displaying certain catalytic characteristics. The gel illustrates the substrate acceptance spectra and selectivities of the different proteins.
Trypanothione reductase (TryR) is a key validated enzyme in the trypanothione‐based redox metabolism of pathogenic trypanosomes and leishmania parasites. This system is absent in humans, being replaced with glutathione and glutathione reductase, and as such offers a target for selective inhibition. As part of a program to discover antiparasitic drugs, the LOPAC1280 library of 1266 compounds was screened against TryR and the top hits evaluated against glutathione reductase and T. brucei parasites. The top hits included a number of known tricyclic neuroleptic drugs along with other new scaffolds for TryR. Three novel druglike hits were identified and SAR studies on one of these using information from the tricyclic neuroleptic agents led to the discovery of a competitive inhibitor (Ki=330 nM ) with an improved potency against T. brucei (EC50=775 nM ).相似文献
Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR+. The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2-nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches. 相似文献
17β‐Hydroxysteroid dehydrogenase (17β‐HSDcl) from the filamentous fungus Curvularia lunata (teleomorph Cochliobolus lunatus) catalyzes NADP(H)‐dependent oxidoreductions of androgens and estrogens. Despite detailed biochemical and structural characterization of 17β‐HSDcl, its physiological function remains unknown. On the basis of amino acid sequence alignment, phylogenetic studies, and the recent identification of the physiological substrates of the homologous MdpC from Aspergillus nidulans and AflM from Aspergillus parasiticus, we propose an anthrahydroquinone as the physiological substrate of 17β‐HSDcl. This is also supported by our analysis of a secondary metabolite biosynthetic gene cluster in C. lunata m118, containing 17β‐HSDcl and ten other genes, including a polyketide synthase probably involved in emodin formation. Chemoenzymatic reduction of emodin by 17β‐HSDcl in the presence of sodium dithionite verified this hypothesis. On the basis of these results, the involvement of a 17β‐HSDcl in the biosynthesis of other anthrahydroquinone‐derived natural products is proposed; hence, 17β‐HSDcl should be more appropriately referred to as a polyhydroxyanthracene reductase (PHAR). 相似文献
Oxidoreductases are attractive biocatalysts that convert achiral substrates into products of higher value, but they are also for the most part dependent on nicotinamide cofactors. Recently, biomimetic nicotinamide derivatives have received attention as less costly alternatives to natural cofactors. However, recycling of biomimetics is still challenging because there are only limited opportunities. Here, we have characterized various biomimetic cofactors with regard to stability and redox potentials to find the best alternative to natural cofactors. Further, the cofactor spectrum of NADH oxidase from Lactobacillus pentosus (LpNox) could be expanded, and the enzymatic activity was also compared to activities with different small‐molecule catalysts. As a result, we succeeded in identifying several strategies for regeneration of oxidized biomimetics. 相似文献