Methyltransferases (MTases) modify a wide range of biomolecules using S-adenosyl-l-methionine (AdoMet) as the cosubstrate. Synthetic AdoMet analogues are powerful tools to site-specifically introduce ...a variety of functional groups and exhibit potential to be converted only by distinct MTases. Extending the size of the substituent at the sulfur/selenium atom provides selectivity among MTases but is insufficient to discriminate between promiscuous MTases. We present a panel of AdoMet analogues differing in the nucleoside moiety (NM-AdoMets). These NM-AdoMets were efficiently produced by a previously uncharacterized methionine adenosyltransferase (MAT) from methionine and ATP analogues, such as ITP and N
-propargyl-ATP. The N
-modification changed the relative activity of three representative MTases up to 13-fold resulting in discrimination of substrates for the methyl transfer and could also be combined with transfer of allyl and propargyl groups.
Abstract
Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Most of these modifications are ...methyl groups, making
S
-adenosyl-
L
-methionine (SAM) a central metabolic hub. Here we show that metabolic labeling with a clickable metabolic precursor of SAM, propargyl-selenohomocysteine (PSH), enables detection and identification of various methylation sites. Propargylated A, C, and G nucleosides form at detectable amounts via intracellular generation of the corresponding SAM analogue. Integration into next generation sequencing enables mapping of
N
6
-methyladenosine (m
6
A) and 5-methylcytidine (m
5
C) sites in mRNA with single nucleotide precision (MePMe-seq). Analysis of the termination profiles can be used to distinguish m
6
A from 2′-
O
-methyladenosine (A
m
) and
N
1-methyladenosine (m
1
A) sites. MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, which was limiting previous methodologies. Metabolic labeling via clickable SAM facilitates the joint evaluation of methylation sites in RNA and potentially DNA and proteins.
Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site‐specific ...inhibition and timed removal. S‐Adenosyl‐L‐methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC‐ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC‐ChMAT at 1.87 Å revealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC‐MATs were compatible with DNA‐ and RNA‐MTases, enabling sequence‐specific modification (“writing”) of plasmid DNA and light‐triggered removal (“erasing”).
S‐Adenosyl‐L‐methionine (AdoMet) analogs provide a way to study and control methyltransferase target sites. Their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered and crystallized the first SAM synthetases (PC‐MATs) able to generate AdoMet analogs with photocaging groups. In combination with DNA‐MTases, sequence‐specific modification (“writing”) of plasmid DNA and light‐triggered removal (“erasing”) is demonstrated.
Methyltransferases (MTases) have become an important tool for site‐specific alkylation and biomolecular labelling. In biocatalytic cascades with methionine adenosyltransferases (MATs), transfer of ...functional moieties has been realized starting from methionine analogues and ATP. However, the widespread use of S‐adenosyl‐l‐methionine (AdoMet) and the abundance of MTases accepting sulfonium centre modifications limit selective modification in mixtures. AdoMet analogues with additional modifications at the nucleoside moiety bear potential for acceptance by specific MTases. Here, we explored the generation of double‐modified AdoMets by an engineered Methanocaldococcus jannaschii MAT (PC‐MjMAT), using 19 ATP analogues in combination with two methionine analogues. This substrate screening was extended to cascade reactions and to MTase competition assays. Our results show that MTase targeting selectivity can be improved by using bulky substituents at the N6 of adenine. The facile access to >10 new AdoMet analogues provides the groundwork for developing MAT‐MTase cascades for orthogonal biomolecular labelling.
An engineered MAT (methionine adenosyltransferase) is capable of generating double‐modified AdoMet analogues (DM‐AdoMets) when provided with non‐natural methionine analogues and nucleoside‐modified ATP analogues. DM‐AdoMets have unique properties and not all MTases (methyltransferases) can easily convert them. This increases selectivity for MTase‐based biomolecular labelling applications, enabling differential MTase targeting wherein multiple MTases are present (i. e., in cell).
Methylation and demethylation of DNA, RNA and proteins constitutes a major regulatory mechanism in epigenetic processes. Investigations would benefit from the ability to install photo‐cleavable ...groups at methyltransferase target sites that block interactions with reader proteins until removed by non‐damaging light in the visible spectrum. Engineered methionine adenosyltransferases (MATs) have been exploited in cascade reactions with methyltransferases (MTases) to modify biomolecules with non‐natural groups, including first evidence for accepting photo‐cleavable groups. We show that an engineered MAT from Methanocaldococcus jannaschii (PC‐MjMAT) is 308‐fold more efficient at converting ortho‐nitrobenzyl‐(ONB)‐homocysteine than the wildtype enzyme. PC‐MjMAT is active over a broad range of temperatures and compatible with MTases from mesophilic organisms. We solved the crystal structures of wildtype and PC‐MjMAT in complex with AdoONB and a red‐shifted derivative thereof. These structures reveal that aromatic stacking interactions within the ligands are key to accommodating the photocaging groups in PC‐MjMAT. The enlargement of the binding pocket eliminates steric clashes to enable AdoMet analogue binding. Importantly, PC‐MjMAT exhibits remarkable activity on methionine analogues with red‐shifted ONB‐derivatives enabling photo‐deprotection of modified DNA by visible light.
An engineered MAT (methionine adenosyltransferase) exhibits remarkable activity on methionine analogues with red‐shifted ortho‐nitrobenzyl (ONB)‐derivatives and is compatible with methyltransferases from thermophilic and mesophilic organisms. This enables photo‐deprotection of modified DNA by visible light. The crystal structure reveals details about substrate accommodation.
Methyltransferases provide excellent specificity in late-stage alkylation of biomolecules. Their dependence on
-adenosyl-L-methionine (SAM) mandates efficient access to SAM analogues for biocatalytic ...applications. We directly compared halide methyltransferase (HMT) and methionine adenosyltransferase (MAT) to access SAM analogues and explored their utility in cascade reactions with NovO for regioselective, late-stage Friedel-Crafts alkylation of a coumarin. The HMT cascade efficiently provided SAM for methylation, while the MAT cascade also supplied high levels of SAM analogues for alkylation reactions.
m6A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3‐METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise ...transcriptome‐wide assignment of m6A sites is of utmost importance. However, m6A does not interfere with Watson–Crick base pairing, making polymerase‐based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl groups can be introduced enzymatically by wild‐type METTL3‐METTL14. Reverse transcription terminated up to 65 % at m6A sites after bioconjugation and purification, hence enabling detection of METTL3‐METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl‐l‐selenohomocysteine and validated different types of known rRNA methylation sites.
Enrich and detect: A chemical biology approach for the precise mapping of RNA methyltransferase target sites in vitro and in cells was developed. A synthetic analogue of the natural cosubstrate or the metabolic prescursor was used to enzymatically introduce bioorthogonal propargyl groups. Subsequent bioconjugation, enrichment, and detection by next‐generation sequencing were demonstrated.
Chemical modification of small molecules is a key step for the development of pharmaceuticals. S‐adenosyl‐l‐methionine (SAM) analogues are used by methyltransferases (MTs) to transfer alkyl, allyl ...and benzyl moieties chemo‐, stereo‐ and regioselectively onto nucleophilic substrates, enabling an enzymatic way for specific derivatisation of a wide range of molecules. l‐Methionine analogues are required for the synthesis of SAM analogues. Most of these are not commercially available. In nature, O‐acetyl‐l‐homoserine sulfhydrolases (OAHS) catalyse the synthesis of l‐methionine from O‐acetyl‐l‐homoserine or l‐homocysteine, and methyl mercaptan. Here, we investigated the substrate scope of ScOAHS from Saccharomyces cerevisiae for the production of l‐methionine analogues from l‐homocysteine and organic thiols. The promiscuous enzyme was used to synthesise nine different l‐methionine analogues with modifications on the thioether residue up to a conversion of 75 %. ScOAHS was combined with an established MT dependent three‐enzyme alkylation cascade, allowing transfer of in total seven moieties onto two MT substrates. For ethylation, conversion was nearly doubled with the new four‐enzyme cascade, indicating a beneficial effect of the in situ production of l‐methionine analogues with ScOAHS.
O‐acetyl‐l‐homoserine sulfhydrolase is used for the production of l‐methionine analogues from l‐homocysteine and off‐the shelf organic thiols. Combined with methyltransferase‐catalysed alkylation cascades, this enables the transfer of a wide range of moieties onto various substrates.
S‐Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM‐dependent enzymes of great interest in ...biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S‐adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one‐pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems’ flexibility is showcased by the transfer of an ethyl group with a cobalamin‐dependent radical SAM MT using S‐adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.
The biomimetic regeneration system for S‐adenosylmethionine (SAM) and SAM analogues presented is based on the salvage of the adenine moiety and in situ supply of d‐ribose and polyphosphate. It is compatible with a broad range of SAM‐dependent enzymes including aminopropyl transferases, and is shown to support ethylation reactions with both conventional and radical SAM methyltransferases.
Ein präziser Kartierungsansatz zur Detektion von Zielstrukturen von RNA‐Methyltransferase wird von A. Rentmeister et al. in ihrer Zuschrift auf S. 6451 ff. beschrieben. Eine bioorthogonale ...Propargylgruppe wird enzymatisch auf RNA übertragen, und die Methyltransferase‐Zielstrukturen können im Anschluss mittels Biokonjugation, Anreicherung sowie Sequenzierungsmethoden der nächsten Generation detektiert werden. Das Bild zeigt die Kartierung eines RNA‐Waldes durch Einführung einer Propargylgruppe zur Unterscheidung zwischen modifizierten und nichtmodifizierten Adenosinen.
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