Uranyl ion‐specific DNAzyme : A DNAzyme (lower strand) cleaves the substrate (upper strand) in the presence of the uranyl ion. The enzyme folds into a bulged three‐way‐junction structure with catalytically important nucleotides residing in the bulge. A highly conserved G?A mismatch is also crucial for the enzyme's activity.
Chemoenzymatic peptide synthesis is potentially the most cost‐efficient technology for the synthesis of short and medium‐sized peptides with some important advantages. For instance, stoichiometric amounts of expensive coupling reagents are not required and racemisation does not occur rendering purification easier compared to chemical peptide synthesis. In this paper, a novel interconversion reaction of peptide C‐terminal α‐carboxamides into primary alkyl esters with alcalase was used to develop a fully enzymatic peptide synthesis strategy. For each elongation step a cost‐efficient amino acid carboxamide building block was used followed by the interconversion of the elongated peptide carboxamide to the corresponding primary alkyl ester. These peptide esters are the starting materials for the next enzymatic peptide elongation step. 相似文献
2‐Methyltetrahydrothiophen‐3‐one ( 3 ) is a volatile compound that plays an important role especially in food and flavour chemistry because it contributes to the aroma of several foodstuffs including wine. Although 3 can be formed by chemical reactions during food preparation, it is also produced by microorganisms. Recent studies with yeasts showed that methionine ( 1 ) is a potential precursor of 3 , but the mechanism of the transformation is unknown. The biosynthetic pathway leading to 3 in the bacterium Chitinophaga Fx7914 was probed. Extensive feeding experiments with differently labelled precursors by using liquid cultures of Chitinophaga Fx7914 were performed. The volatiles released by the bacterium were collected by using a closed loop stripping apparatus (CLSA) and analysed by GC–MS. The observed incorporation pattern of the precursors into 3 led to the elucidation of the biosynthetic pathway. One part of the compound 2 originates from homocysteine ( 15 ), which is transformed into 3‐mercaptopropanal ( 17 ). The second biosynthetic building block is pyruvate ( 14 ). An acyloin‐forming reaction furnishes the key intermediate 21 , which cyclises intramolecularly to a diol. Dehydration followed by tautomerisation lead to the cyclic ketone 3 , which is produced by the bacterium in racemic form.相似文献
The most commonly employed glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position in potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a qualitative mass spectrometry–based glycosidase assay amenable to high‐throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high‐throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved through the observation of an appropriate decrease in mass between a potential sugar substrate and its corresponding product by electrospray ionization mass spectrometry (ESI‐MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus. 相似文献
The EI‐MS fragmentation mechanism of the bacterial sesquiterpene epi‐isozizaene was investigated through enzymatic conversion of all 15 synthetic (13C1)FPP isotopomers with the epi‐isozizaene synthase from Streptomyces albus and GC‐MS and GC‐QTOF analysis including MS‐MS. A systematic method, which we wish to call position‐specific mass shift analysis, for the identification of the full set of fragmentation reactions was developed. 相似文献
Intrinsically disordered regions (IDRs) are preferred sites for post‐translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase–phosphatase networks. Here we used real‐time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the “unique domain” of c‐Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin‐dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP‐dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK‐dependent sites, thus suggesting indirect effects of kinase–phosphatase networks. This study provides a proof‐of‐concept of the use of real‐time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains. 相似文献
Chemoenzymatic peptide synthesis is a rapidly developing technology for cost effective peptide production on a large scale. As an alternative to the traditional C→N strategy, which employs expensive N‐protected building blocks in each step, we have investigated an N→C extension route that is based on activation of a peptide C‐terminal amide protecting group to the corresponding methyl ester. We found that this conversion is efficiently catalysed by Stenotrophomonas maltophilia peptide amidase in neat organic media. The system excludes the possibility of internal peptide cleavage as the enzyme lacks intrinsic protease activity. The produced peptide methyl ester was used for peptide chain extension in a kinetically controlled reaction by a thermostable protease.
A dual maleimide (DuMal) tagging method has been developed for both relative and absolute quantitation of cysteine‐containing peptides (CCPs) in combination with MALDI‐TOF mass spectrometry. A pair of maleimides with minimal differences in their chemical structures, N‐methylmaleimide and Nethylmaleimide, have been chosen to allow for the rapid (≈minutes) tagging of CCPs in the Michael addition reaction with high efficiency. It has been validated that the DuMal tagging technique is sensitive and reliable in the quantitative analysis of CCPs. Absolute quantitation of CCPs can be achieved with a detection limit as low as 7.3 nm . Relative quantitation of CCPs can be performed in various sample mixtures with consistent results (coefficient of variation <5 %). The DuMal tagging technique provides a sensitive and accurate approach for the quantitation of biomolecules containing thiol reactive sites; thus it is promising for protein detection, disease diagnosis, and biomarker discovery associated with post‐translational modifications of cysteines. 相似文献
We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody–peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody–peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody–peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed. 相似文献