A sensitive mass spectrum assay to characterize engineered methionine adenosyltransferases with S-alkyl methionine analogues as substrates

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 (SAAM) 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-alkyl-thioadenosines, followed by HPLC/MS/MS quantification. With this assay, 40 human MAT mutants were evaluated against seven SAAM 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 towards the bulky SAAM 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 SAAM.

Chemical and Biochemical Perspectives of Protein Lysine Methylation

“…(98, 99) Upon the completion of the transfer reaction, PKMTs release the methylated product and a byproduct S -adenosylhomocysteine (SAH) for another catalytic cycle. (19, 100) As detailed below, the structures of many PKMTs have been elegantly tuned to promote the stepwise catalysis.…”

“…(21, 270) Human MAT2A I117A variant was shown to be the so-far most efficient enzyme to process bulky S -alkyl methionine analogs into the corresponding SAM analogs. (19, 270) With G9a and GLP1 as examples, we showed that ( E )-hex-2-en-5-ynyl homocysteine (Hey-methionine analog) can be processed by the I117A variant of human MAT2A into the corresponding Hey-SAM. (270) In the presence of the BPPP-feasible G9a and GLP1 mutants (Y1154A of G9a and Y1211A of GLP1), histone H3 and other chromatin targets of G9a and GLP1 can be efficiently labeled.…”

“…Most biological methylation reactions are catalyzed by methyltransferases with SAM as the methyl donor (Figure 1). (18, 19) Second only to adenosine triphosphate (ATP), SAM is one of the most commonly used enzyme cofactors. (18, 20) Across living organisms, SAM is produced by SAM synthases (also named as S -methionine adenosyltransferases, MATs) with L-methionine and ATP as substrates (Figure 1).…”

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation

“…In 2013, F. Truong [11] used a chemical procedure to synthesise Sahc and proved its incorporation into a GFP variant upon feeding Met-auxotrophic Escherichia coli cells. In addition to its translational activity, Sahc is also metabolically active, since it serves as a substrate for Met-adenosyltransferases (MATs) which are crucial enzymes in the biosynthesis of the central metabolite S-adenosylmethionine (SAM) [12,13,14,15]. Met is also the standard starting residue in ribosomal translation, although 60% of these residues are removed by N-terminal processing in E. coli [16].…”

Nucleoside-modified AdoMet analogues for differential methyltransferase targeting

“…1 (A) Previous work showed that methionine analogues and ATP can be converted by methionine adenosyltransferase (MAT) to AdoMet analogues. [23][24][25][26]30 These were used as cosubstrates in alkylation reactions catalyzed by promiscuous methyltransferases (MTases). (B) In this work, we show that methionine or analogues together with ATP analogues can be converted to nucleoside-modified AdoMet analogues (NM-AdoMets) allowing for selectivity among different MTases.…”