Elmar G. Weinhold

Structure of the N6-adenine DNA methyltransferase M.TaqI in complex with DNA and a cofactor analog.

The 2.0 Å crystal structure of the N6-adenine DNA methyltransferase M•TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5′-TCGA-3′, the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand π-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.

Enzymatic Site‐Specific Functionalization of Protein Methyltransferase Substrates with Alkynes for Click Labeling

Posttranslational modifications of proteins are key to essentially all regulatory processes in cells. Many different modifications, including methylation, have been described for core histones, the protein components of nucleosomes. The modifications occur preferentially on the N-terminal tails and are thought to control the interaction with proteins associated with the regulation of chromatin structure and gene transcription. Recent studies have demonstrated that methylation of the side chains of lysine and arginine residues of core histones are associated with specific functional states of promoters. For example, methylation of histone H3 at lysine 9 (H3K9) is a negative mark for gene transcription, and trimethylation of histone H3 at lysine 4 (H3K4) is a marker for transcribed promoters. Methylation at H3K4 is interconnected with other histone modifications, including dimethylation of histone H3 at arginine 2 (H3R2), a transcriptionally negative mark which inhibits methylation of H3K4. 7] Protein methyltransferases (MTases) transfer the activated methyl group from the cofactor S-adenosyl-l-methionine (AdoMet or SAM) mainly to lysine and arginine side chains in their protein substrates. These enzymes are often sequence-specific; for example, mixed-lineage leukaemia (MLL) histone MTase complexes trimethylate H3K4. Methylation of lysine residues is a dynamic, reversible modification involving MTases and demethylases. The main protein MTase substrates described are core histones and a few proteins associated with gene transcription. Comprehensive analyses of MTase substrates are lacking, at least in part because the methyl group is a poor reporter. Antibodies seem to recognize methylated amino acids only in a context-dependent manner, that is, in combination with the underlying peptide sequence. Therefore we thought to develop alternative methods to identify MTase substrates. Recently, we reported on synthetic double-activated AdoMet analogues with allylic and propargylic methyl group replacements for site-specific DNA modification by DNA MTases. Such analogues also function as cofactors for small molecule MTases. Compared to aziridinium-based AdoMet analogues, these cofactors have the advantage that strong product inhibitors are not formed during the MTasecatalyzed reaction. When an amino function was appended to the propargylic side chain, it was possible to couple Nhydroxysuccinimde (NHS)-activated reporters to the modified DNA in a second step. Since introduction of amino groups is generally not productive for the analysis of proteins, we designed the new AdoMet-based cofactor AdoEnYn (1; Scheme 1), in which

Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling

This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNAPhe. LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition click chemistry, producing site-specifically labeled RNA whose suitability for single molecule fluorescence experiments was verified in fluorescence correlation spectroscopy experiments.

Synthesis of S-adenosyl-L-methionine analogs and their use for sequence-specific transalkylation of DNA by methyltransferases

Here we describe a one-step synthetic procedure for the preparation of S-adenosyl-L-methionine (AdoMet) analogs with extended carbon chains replacing the methyl group. These AdoMet analogs function as efficient cofactors for DNA methyltransferases (MTases), and we provide a protocol for sequence-specific transfer of extended side chains from these AdoMet analogs to DNA by DNA MTases. Direct chemoselective allylation or propargylation of S-adenosyl-L-homocysteine (AdoHcy) at sulfur is achieved under the acidic conditions needed to protect other nucleophilic positions in AdoHcy. The unsaturated bonds in β position to the sulfonium center of the resulting AdoMet analogs are designed to stabilize the transition state formed upon DNA MTase-catalyzed nucleophilic attack at the carbon next to the sulfonium center and lead to efficient transfer of the extended side chains to DNA. Using these protocols, sequence-specific functionalized DNA can be obtained within one to two weeks.

A Selenium‐Based Click AdoMet Analogue for Versatile Substrate Labeling with Wild‐Type Protein Methyltransferases

Protein methylation is catalyzed by S‐adenosyl‐L‐methionine‐dependent protein methyltransferases (MTases), and this posttranslational modification serves diverse cellular functions. Some MTases seem to exhibit broad substrate specificities and comprehensive methods for target profiling are needed. Here we report the synthesis of a new AdoMet analogue for enzymatic transfer of a small propargyl group and labeling of modified proteins through copper‐catalyzed azide–alkyne cycloaddition (CuAAC). Replacement of sulfur by selenium strongly enhanced the stability of the progargylic cofactor, leading, in combination with better activation by the selenonium center, to higher enzymatic reactivity. A broad spectrum of wild‐type protein MTases acting on lysine, arginine, and glutamine residues accept this cofactor and modified substrates can be efficiently labeled by CuAAC click chemistry.

Synthesis of S‐Adenosyl‐L‐homocysteine Capture Compounds for Selective Photoinduced Isolation of Methyltransferases

Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity‐based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S‐adenosyl‐L‐methionine (SAM or AdoMet)‐dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S‐adenosyl‐L‐homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase–cofactor complexes.

Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases

Biophysical and mechanistic investigation of RNA function requires site-specific incorporation of spectroscopic and chemical probes, which is difficult to achieve using current technologies. We have in vitro reconstituted a functional box C/D small ribonucleoprotein RNA 2′-O-methyltransferase (C/D RNP) from the thermophilic archaeon Pyrococcus abyssi and demonstrated its ability to transfer a prop-2-ynyl group from a synthetic cofactor analog to a series of preselected target sites in model tRNA and pre-mRNA molecules. Target selection of the RNP was programmed by changing a dodecanucleotide guide sequence in a 64-nt C/D guide RNA leading to efficient derivatization of three out of four new targets in each RNA substrate. We also show that the transferred terminal alkyne can be further appended with a fluorophore using a bioorthogonal azide-alkyne 1,3-cycloaddition (click) reaction. The described approach for the first time permits synthetically tunable sequence-specific labeling of RNA with single-nucleotide precision.

Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA)

A new concept for sequence‐specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S‐adenosyl‐L‐methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase‐catalyzed sequence‐specific coupling with DNA. Sequence‐specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups.

Coupling of a Nucleoside with DNA by a Methyltransferase

How to outwit a methyltransferase: Methyltransferases (Mtases) catalyze the transfer of the activated methyl group from the cofactor S-adenosyl-L-methionine (1) to acceptors R within a large variety of biomolecules. Through the use of the cofactor analogue 2 a whole nucleoside was coupled to DNA in a Mtase-catalyzed reaction.

Efficient Synthesis of S‐Adenosyl‐L‐Homocysteine Natural Product Analogues and Their Use to Elucidate the Structural Determinant for Cofactor Binding of the DNA Methyltransferase M·HhaI

5′-Acetylthio-5′-deoxy-2′,3′-O-isopropylideneadenosine (8) was directly prepared from commercially available 2′,3′-O-isopropylideneadenosine (7) and thioacetic acid under Mitsunobu conditions in almost quantitative yield. In situ cleavage of the acetylthio function of 8 followed by coupling with different alkyl bromides proceeded with high yields. Deprotection of the obtained 5′-thionucleosides yielded the S-adenosyl-L-homocysteine analogues decarboxylated AdoHcy (11), deaminated AdoHcy (14) and 5′-[3-(cyano)propylthio]-5′-deoxyadenosine (16) in good overall yields. Direct deprotection of the thionucleoside 8 delivered 5′-thio-5′-deoxyadenosine (18) in excellent yield. In addition, binding constants of these AdoHcy analogues and the DNA methyltransferase M·HhaI were determined in a fluorescence assay.