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• W2055269317 endingPage "19" @default.
• W2055269317 startingPage "11" @default.
• W2055269317 abstract "Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-l-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAMs) into the SAM analogues with engineered MATs. Here we describe a general sensitive activity assay for engineered MATs by converting the reaction products into S-alkylthioadenosines, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification. With this assay, 40 human MAT mutants were evaluated against 7 SAAMs as potential substrates. The structure-activity relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower Km value in contrast to native MATs), the gained activity toward the bulky SAAMs stems from their ability to maintain the desired linear SN2 transition state (reflected by higher kcat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAMs." @default.
• W2055269317 created "2016-06-24" @default.
• W2055269317 creator A5040396327 @default.
• W2055269317 creator A5053032258 @default.
• W2055269317 creator A5088701116 @default.
• W2055269317 date "2014-04-01" @default.
• W2055269317 modified "2023-10-17" @default.
• W2055269317 title "A sensitive mass spectrum assay to characterize engineered methionine adenosyltransferases with S-alkyl methionine analogues as substrates" @default.
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• W2055269317 doi "https://doi.org/10.1016/j.ab.2013.12.026" @default.
• W2055269317 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3947680" @default.
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• W2055269317 hasPublicationYear "2014" @default.
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