SAM analogues & protein engineering
By developing and applying S-Adenosyl-L-methionine (SAM) analogues, our research investigates biochemical processes, with a particular focus on DNA and RNA methylation. We engineer methionine adenosyltransferase (MAT) enzymes to produce SAM analogues with extended side chains and photocleavable groups, allowing for the selective labeling, enrichment, and controlled activation of biomolecules. These innovative SAM analogues enable precise modifications and advanced studies of methyltransferase targets and their roles in cellular regulation.
S-Adenosyl-L-methionine (SAM or AdoMet) is the second most abundant cofactor in the cell after ATP and the primary methyl donor in nature. SAM plays a pivotal role in various biochemical processes, including DNA and RNA methylation. We have developed and applied a number of so-called AdoMet analogues (Figure 1) with remarkably extended side chains at the sulfonium center.
Figure 1: Chemoenzymatic Production of SAM analogues. Methionine derivatives can be created through the reductive alkylation of homocystine. SAM analogues can be formed by combining methionine analogues with ATP using MATs. For the chemical synthesis of SAM analogues, SAH can be alkylated directly with alkyl triflates or alkyl bromides, yielding a mix of stereoisomers.
In combination with a promiscuous methyltransferases, we used AdoMet analogues for the transfer of biorthogonal groups to label and enrich biomolecules via their methyltransferase target sites (Figure 2). We also developed AdoMet analogues with photocleavable groups to block biochemical functions of DNA and RNA and then reactivate them by irradiation with light.
In our group, we perform the chemical synthesis of AdoMet analogues but also follow the biosynthetic route (Figure 1). In nature, the enzyme MAT (Methionine adenosyltransferase) generates SAM from methionine and ATP.
Figure 2: General chemo-enzymatic approaches to RNA cap modifications and their structures. Scheme illustrating co- and post-transcriptional modification as approaches to generate cap-modified RNAs using MTases.
We have engineered MAT enzymes to accept methionine analogues (1, 2). The crystal structure revealed that AdoONB is accomodated by pi-stacking of the ONB ring to the adenine part of the molecule. The engineered MAT (PC-MjMAT) shows a broad substrate spectrum and can be used to generate a range of AdoMet analogues with benzylic groups (3).
Figure 3: Engineering of MAT from Methanocaldococcus jannaschii by Peters et al. leads to the SAM analog promiscuous MAT variant PC-MjMAT (L147A/I351A).The overall structure of PC-MjMAT (PDB: 7P84) shows an open/closed gating loop. Close-up view of the gating loop blocking access to the active pocket. Direct view of amino acid substitution L147A and I351A together with the substrate ONBHcy within the active pocket. Due to the exchange of leucine with alanine, the substrate ONBHcy undergoes fewer steric interactions.
Enzymatic generation of double-modified SAM analogues
Methionine adenosyltransferases (MATs) produce S-adenosyl-L-methionine (SAM) from methionine and ATP. SAM serves as a cosubstrate for most methyltransferases (MT), however many of them are promiscuous with regard to the methyl (or alkyl or benzyl) group at the sulfonium centre. The widespread occurrence of MTs prevents the use of SAM analogues with extended moieties at the sulfonium centre for studying individual MTs or labeling biomolecules selectively in cellular systems. We therefore develop double-modified SAM analogues with modifications at the sulfonium centre and the adenosine part of SAM (Figure 4). The combined modifications shall prevent the conversion by wildtype MTs.
Figure 4: Cascade reactions and substrate analogues. A) Methionine adenosyltransferase (MAT) for enzymatic generation of SAM and SAM analogues. The modification at different sites of SAM alters how well the SAM analogues are converted by different MT, providing a way to achieve selective modification with non-natural residues in complex mixtures. B) Schematic illustration of SAM binding pocket of MTs. R1 (blue), R2 (green), and R3 (red) indicate residues at the nucleobase, the ribose, or the sulfonium (S)/selenium (Se) centre (X), respectively. C) Heat maps showing activity of PC-MjMAT on a panel of substrates indicated above. Nucleoside-modified ATP analogues (a1-, b1-, c1-, d2-, e2-, e1-, f1-, i3-, g2-, g1-, i2-, h1-, i1-, j1-, k1-, l1-, m1-, n1-, and o1-triphosphates) were reacted with y-Hcy (ONB-Hcy) or z-SeHcy (propargyl-selenohomocysteine). Activity is calculated based on the conversion of methionine analogue and given as percentage in each tile of the heatmap. Colour coding from light-yellow to dark-red indicates conversion as indicated on the left. The zero (0 h) and end (6 h) time point activity assays were performed as three independent experiments. C) Heat map showing activity of NovO, RnCOMT-var (M40A-Y200L), and GlaTgs2-var (V34A) in cascade with PC-MjMAT on a panel of double-modified (i.e., nucleoside- and sulfonium/selenium centre-modified) SAM analogues. Activity is calculated based on the conversion of MTase substrate and given as percentage in each tile of the heatmap. THPC (4,5,7-trihydroxy-3-phenylcoumarin), DHBAL (3,4-dihydroxybenzaldehyde), and the 5′ cap analogue m7GpppA were used as substrates of NovO, RnCOMT-var, and GlaTgs-var, respectively. Colour coding from light-yellow to dark-red indicates conversion as indicated on the right. The zero (0 h) and end (6 h or 8 h) time point activity assays were performed as two independent experiments. “n.d.” (not determined). (Figure modified from (3)).
References
1. Michailidou, F.; Klöcker, N.; Cornelissen, N. V.; Singh, R.; Peters, A.;
Ovcharenko, A.; Kümmel, D.; Rentmeister, A.
Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with
Photocaging Groups and Reversible DNA Modification in Cascade Reactions.
Angewandte Chemie International Edition 2020, 60 (1), 480–485.
2. Peters, A.; Herrmann, E.; Cornelissen, N. V.; Klöcker, N.; Kümmel, D.; Rentmeister, A.
Visible‐Light Removable Photocaging Groups Accepted by MjMAT Variant:
Structural Basis and Compatibility with DNA and RNA Methyltransferases.
ChemBioChem 2021, 23 (1).
3. Cornelissen, N. V.; Mineikaitė, R.; Erguven, M.; Muthmann, N.; Peters, A.; Bartels, A.;
Rentmeister, A.
Post-Synthetic Benzylation of the MRNA 5′ Cap via Enzymatic Cascade Reactions.
Science 2023, 14 (39), 10962–10970.