534-30-5Relevant articles and documents
Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay
Banco, Michael T.,Mishra, Vidhi,Greeley, Samantha C.,Ronning, Donald R.
, p. 1740 - 1747 (2018)
S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) are an essential superfamily of enzymes that catalyze the transfer of a methyl group to several biomolecules. Alterations in the methylation of cellular components crucially impact vital biological processes, making MTases attractive drug targets for treating infectious diseases and diseases caused by overactive human-encoded MTases. Several methods have been developed for monitoring the activity of MTases, but most MTase assays have inherent limitations or are not amenable for high-throughput screening. We describe a universal, competitive fluorescence polarization (FP) assay that directly measures the production of S-adenosylhomocysteine (AdoHcy) from MTases. Our developed assay monitors the generation of AdoHcy by displacing a fluorescently labeled AdoHcy molecule complexed to a catalytically inert 5′-methylthioadenosine nucleosidase (MTAN-D198N) variant performed in a mix-and-read format. Producing the fluorescently labeled molecule involves a one-pot synthesis by combining AdoHcy with an amine-reactive rhodamine derivative, which possesses a Kd value of 11.3 ± 0.7 nM to MTAN-D198N. The developed competitive FP assay expresses a limit of detection for AdoHcy of 6 nM and exhibits a 34-fold preference to AdoHcy in comparison to AdoMet. We demonstrate the utility of the developed assay by performing a pilot screen with the NIH Clinical Collection as well as determining the kinetic parameters of l-histidine methylation for EgtD from Mycobacterium tuberculosis. Additionally, the developed assay is applicable to other AdoMet-dependent and ATP-dependent enzymes by detecting various adenosine-containing molecules including 5′-methylthioadenosine, AMP, and ADP.
Identification of the main intermediate precursor of L-ergothioneine biosynthesis in human biological specimens
Sotgia, Salvatore,Mangoni, Arduino A.,Forteschi, Mauro,Murphy, Rhys B.,Elliot, David,Sotgiu, Elisabetta,Pintus, Gianfranco,Carru, Ciriaco,Zinellu, Angelo
, (2016)
A capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) has been used to make a qualitative determination of hercynine - the main precursor of L-ergothioneine biosynthesis - in some key human biological specimens, such as urine, whole blood, plasma, and saliva. From semiquantitative analysis results, the highest concentrations of hercynine were detected in saliva and whole blood, whereas much lower concentrations were measured in urine and plasma. Whole blood was the biological matrix with the highest concentration of L-ergothioneine followed by plasma, saliva, and urine. The antioxidant effects attributed to L-ergothioneine, along with its peculiar antioxidant mechanism, offer a possible explanation for the presence of the hercynine, as well as its concentration, in the considered biological matrices.
Development of an LC–tandem mass spectrometry method for the quantitative analysis of hercynine in human whole blood
Sotgia, Salvatore,Murphy, Rhys B.,Zinellu, Angelo,Elliot, David,Paliogiannis, Panagiotis,Pinna, Gerard Aimè,Carru, Ciriaco,Mangoni, Arduino A.
supporting information, (2019/01/03)
Given that the peculiar redox behavior of ergothioneine involves a rapid regeneration process, the measurement of its precursor and redox metabolite hercynine could be particularly useful in assessing its role in oxidative stress or other biological processes. Thus, a LC-MS/MS method for the determination of hercynine concentrations in whole blood was developed. After lysis of red blood cells by cold water, samples were filtered on micro concentrators at a controlled temperature of 4 ?C. The clear filtered fluid was then treated with diethylpyrocarbonate to derivatize hercynine for the analysis by LC-MS/MS. The derivatized analyte was isocratically separated as a carbethoxy derivative on a C18 column with a mobile phase of an aqueous 0.1% v/v formic acid and acetonitrile (95:5). Effluents were monitored by MRM transitions at m/z 270.28→95 and 273.21→95 for hercynine and its deuterated counterpart, respectively. No cross-talk between MRM transitions was observed and a good linearity was found within a range of 35–1120 nmol/L. The LOD and LOQ were, respectively, 10.30 and 31.21 nmol/L with an intraday and intermediate precision below 7%. The average hercynine concentration in whole blood from 30 healthy male volunteers (aged 77 ± 12 years) was 178.5 ± 118.1 nmol/L. Overall, the method is easy to perform, allowing a rapid and accurate assessment of whole blood concentrations of hercynine.
