Institut für Organische Chemie
AK Weinhold

Enzymatically Incorporated Genomic Tags for Optical Mapping of DNA-Binding Proteins

Kim, Soohong; Gottfried, Anna; Lin, Ron R.; Dertinger, Thomas; Kim, Andrew S.; Chung, Sangyoon; Colyer, Ryan A.; Weinhold, Elmar; Weiss, Shimon; Ebenstein, Yuval; Angew. Chem. Int. Ed. 2012, 51(15), 3578-3581.

Sequence-specific methyltransferase-induced labeling of DNA (SMILing DNA) creates a fluorescence pattern of the T7 bacteriophage genome (see picture). The pattern is visualized as a linear optical barcode showing the genomic location of individual RNA polymerases bound to the DNA. The precision of the measurement presents new opportunities for contextual genomic research on the single-molecule level.

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

Willnow, Sophie; Martin, Michael; Lüscher, Bernhard; Weinhold, Elmar; ChemBioChem 2012, 13(8), 1167-1173.

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.

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

Tomkuvienė, Miglė; Clouet-d'Orval, Béatrice; Černiauskas, Ignas; Weinhold, Elmar; Klimašauskas, Saulius; Nucl. Acids Res. 2012, 40(14), 6765-73.

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.

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

Motorin, Yuri; Burhenne, Jürgen; Teimer, Roman; Koynov, Kaloian; Willnow, Sophie; Weinhold, Elmar; Helm, Mark; Nucl. Acids Res. 2011, 39(5), 1943-1952.

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.

Sequence-specific covalent labelling of DNA

Gottfried, Anna; Weinhold, Elmar; Biochem. Soc. Trans. 2011, 39, 623-628.

Sequence-specific DNA modification is of significance for applications in bio- and nano-technology, medical diagnostics and fundamental life sciences research. Preferentially, labelling should be performed covalently, which avoids doubts about label dissociation from the DNA under various conditions. Several methods to label native DNA have been developed in the last two decades. Triple-helix-forming oligodeoxynucleotides and hairpin polyamides that bind DNA sequences specifically in the major and minor groove respectively were used as targeting devices for subsequent covalent labelling. In addition, enzyme-directed labelling approaches utilizing nicking endonucleases in combination with DNA polymerases or DNA methyltransferases have been employed. This review summarizes various techniques useful for functionalization of long native DNA.

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

Peters, Wibke; Willnow, Sophie; Duisken, Mike; Kleine, Henning; Macherey, Thomas; Duncan, Kelly E.; Litchfield, David W.; Lüscher, Bernhard; Weinhold, Elmar; Angew. Chem. 2010, 49(30), 5170-5173.

Protein methylation is an important posttranslational modification. Because the methyl group is a poor reporter group, new methods are needed to analyze methyltransferase substrates. A S-adenosyl-L-methionine-based cofactor was synthesized and used for the site-specific functionalization of proteins with alkynes by methyltransferases (first step) and subsequent labeling through CuAAC click chemistry (second step).

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

Dalhoff, Christian; Hüben, Michael; Lenz, Thomas; Poot, Peter; Nordhoff, Eckhard; Köster, Hubert; Weinhold, Elmar; ChemBioChem 2010, 11(2), 256-265.

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.

Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

Van der Gun, Bernardina T. F.; Maluszynska-Hoffman, Maria; Kiss, Antal; Arendzen, Alice J.; Ruiters, Marcel H. J.; McLaughlin, Pamela M. J.; Weinhold, Elmar; Rots, Marianne G.; Bioconjugate Chem. 2010, 21(7), 1239-1245.

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO−C141S) specifically designed for the EpCAM gene. Reporter plasmids encoding the green fluorescent protein under control of different EpCAM promoter fragments were treated with the TFO−C141S conjugate to determine the specificity of targeted DNA methylation in the context of a functional EpCAM promoter. Treatment of the plasmids with TFO−C141S resulted in efficient and specific methylation of the targeted CpG located directly downstream of the triple helix forming site (TFS). No background DNA methylation was observed neither in a 700 bp region of the EpCAM promoter nor in a 400 bp region of the reporter gene downstream of the TFS. Methylation of the target CpG did not have a detectable effect on promoter activity. This study shows that the combination of a specific TFO and a reduced activity methyltransferase variant can be used to target DNA methylation to predetermined sites with high specificity, allowing determination of crucial CpGs for promoter activity.

Selective recognition of pyrimidine-pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators

Bahr, Matthias; Gabelica, Valerie; Granzhan, Anton; Teulade-Fichou, Marie-Paule; Weinhold, Elmar; Nucleic Acids Res. 2008, 36(15), 5000-5012.

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidinepyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance-constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes.

6-Thioguanine in DNA as CD-spectroscopic probe to study local structural changes upon protein binding

Repges, Rudolph; Beuck, Christine; Weinhold, Elmar; Raabe, Gerhard; Fleischhauer, Jorg; Chirality 2008, 20(9), 978-984.

A combination of experimental and theoretical circular dichroism (CD) spectroscopy was used to study local deformations of DNA caused by binding of the base flipping DNA methyltransferase M.TaqI. To selectively study the structural changes within the DNA, we replaced single guanine residues at six different positions in duplex DNA with 6-thioguanine (S(6)G), which absorbs at 342 nm where unmodified DNA and the enzyme are transparent. The shape and the transition wavelength of a CD signal around 340 nm in the spectra of the free DNA and the M.TaqI-bound DNA were found to depend on the position of the S(6)G probe. Theoretical rotational strengths were calculated employing the matrix method which is frequently used to model the CD of large biomolecules. The only chromophores in these calculations were the nucleic acid bases. Comparison of the measured and the calculated CD spectra showed that the applied computational method qualitatively reproduces the dominant band observed around 340 nm in all cases. From our results we conclude that the spectral changes observed upon binding of the enzyme to the DNA are indeed predominantly due to structural changes within the DNA and not to other effects caused by the presence of the enzyme.

Persistent down-regulation of the pancarcinoma-associated Epithelial Cell Adhesion Molecule via active intranuclear methylation

Van der Gun, Bermadina T.F.; Wasserkort, Reinold; Monami, Amelie; Jeltsch, Albert; Rasko, Tamits; Slaska-Kiss, Krystyna; Cortese, Rene; Rots, Marianne G.; de Leij, Lou F.M.H.; Ruiters Marcel H.J.; Kiss, Antal; Weinhold, Elmar; McLaughlin, Pamela M.J.; Int. J. Cancer 2008, 123(2), 484-489.

The epithelial cell adhesion molecule (EpCAM) is expressed at high levels on the surface of most carcinoma cells. SiRNA silencing of EpCAM expression leads to reduced metastatic potential of tumor cells demonstrating its importance in oncogenesis and tumor progression. However, siRNA therapy requires either sequential delivery or integration into the host cell genome. Hence we set out to explore a more definite form to influence EpCAM gene expression. The mechanisms underlying the transcriptional activation of the EpCAM gene, both in normal epithelial tissue as well as in carcinogenesis, are poorly understood. We show that DNA methylation plays a crucial role in EpCAM expression, and moreover, active silencing of endogenous EpCAM via methylation of the EpCAM promoter results in a persistent downregulation of EpCAM expression. In a panel of carcinoma derived cell lines, bisulfite analyses showed a correlation between the methylation status of the EpCAM promoter and EpCAM expression. Treatment of EpCAM-negative cell lines with a demethylating agent induced EpCAM expression, both on mRNA and protein level, and caused upregulation of EpCAM expression in an EpCAM-positive cell line. After delivery of the DNA methyltransferase M.SssI into EpCAM-positive ovarian carcinoma cells, methylation of the EpCAM promoter resulted in silencing of EpCAM expression. SiRNA-mediated silencing remained for 4 days, after which EpCAM re-expression increased in time, while M.SssI-mediated downregulation of EpCAM maintained through successive cell divisions as the repression persisted for at least 17 days. This is the first study showing that active DNA methylation leads to sustained silencing of endogenous EpCAM expression.

Enzyme-directed positioning of nanoparticles on large DNA templates

Braun, Gary; Diechrierow, Michael; Wilkinson, Stephanie; Schmidt, Falk; Hüben, Michael; Weinhold, Elmar; Reich, Norbert O.; Bioconjugate Chem. 2008, 19(2), 476-479.

A method to position nanoparticles onto DNA with high resolution using an enzyme-based approach is described. This provides a convenient route to assemble multiple nanoparticles (e.g., An and CdSe) to specific positions with a high level of control and expandability to more complex assemblies. Atomic force microscopy is used to analyze the nanostructures, which have potential interest for biosensor, optical waveguide, molecular electronics, and energy transfer studies.

Molecular scale architecture: engineered three- and four-way junctions

Wilkinson, Stephanie; Diechtierow, Michael; Estabrook, R. August; Schmidt, Falk; Hüben, Michael; Weinhold, Elmar, Bioconjugate Chem. 2008, 19(2), 470-475.

Biomolecular self-assembly provides a basis for the bottom-up construction of useful and diverse nanoscale architectures. DNA is commonly used to create these assemblies and is often exploited as a lattice or an array. Although geometrically rigid and highly predictable, these sheets of repetitive constructs often lack the ability to be enzymatically manipulated or elongated by standard biochemical techniques. Here, we describe two approaches for. the construction of position-controlled, molecular-scale, discrete, three- and four-way DNA junctions. The first approach for constructing these junctions relies on the use of nonmigrating cruciforms generated from synthetic oligonucleotides to which large, biologically generated, double-stranded DNA segments are enzymatically ligated. The second approach utilitizes the DNA methyltransferase-based SMILing (sequence-specific methyltransferase-induced labeling of DNA) method to site-specifically incorporate a biotin within biologically derived DNA. Streptavidin is then used to form junctions between unique DNA strands. The resultant assemblies have precise and predetermined connections with lengths that can be varied by enzymatic or hybridization techniques, or geometrically controlled with standard DNA functionalization methods. These junctions are positioned with single nucleotide resolution on large, micrometer-length templates. Both approaches generate DNA assemblies which are fully compatible with standard recombinant methods and thus provide a novel basis for nanoengineering applications.

Sequence-specific Methyltransferase-Inducd Labelling (SMILing) of plasmid DNA for studying cell transfection

Schmidt, Falk H.-G.; Hüben, Michael; Gider, Basar; Renault, Francois; Teulade-Fichou, Marie-Paule; Weinhold, Elmar, Bioorg. Med. Chem. 2008, 16(1), 40-48.

Plasmid DNA (pUC19 and pBR322) was sequence-specifically, covalently labelled with Cy3 fluorophores using a newly synthesised N-adenosylaziridine cofactor and the DNA methyltransferase M.TaqI. The fluorescently labelled plasmids were used for transfection of mammalian cells and their intracellular distribution was visualised by epifluorescence and confocal fluorescence microscopy. Although these prokaryotic plasmids do not contain nuclear import sequences, translocation into the nuclei was observed.

Serum insensitive, intranuclear protein delivery by the multipurpose cationic lipid Saint-2

Van der Gun, Bermadina T.F.; Monami, Amelie; Laarman, Sven; Rasko, Tamas; Slaska-Kiss, Krystyna; Weinhold, Elmar; Wasserkort, Reinhold; de Leij, Lou F.M.H; Ruiters, Marcel H.J.; Kiss, Antal; McLoughlin, Pamela M.J., Journal of Controlled Release 2007, 123(3), 228-238.

Cationic liposomal compounds are widely used to introduce DNA and siRNA into viable cells, but none of these compounds are also capable of introducing proteins. Here we describe the use of a cationic amphiphilic lipid SAINT-2:DOPE for the efficient delivery of proteins into cells (profection). Labeling studies demonstrated equal delivery efficiency for protein as for DNA and siRNA. Moreover, proteins complexed with SAINT-2:DOPE were successfully delivered, irrespective of the presence of serum, and the profection efficiency was not influenced by the size or the charge of the protein:cationic liposomal complex. Using beta-galactosidase as a reporter protein, enzymatic activity was detected in up to 98% of the adherent cells, up to 83% of the suspension cells and up to 70% of the primary cells after profection. A delivered antibody was detected in the cytoplasm for up to 7 days after profection. Delivery of the methyltransferase M.SssI resulted in DNA methylation, leading to a decrease in E-cadherin expression. The lipid-mediated multipurpose transport system reported here can introduce proteins into the cell with an equal delivery efficiency as for nucleotides. Delivery is irrespective of the presence of serum, and the protein can exert its function both in the cytoplasm and in the nucleus. Furthermore, DNA methylation by M.SssI delivery as a novel tool for gene silencing has potential applications in basic research and therapy.

DNA labelling topologies for monitoring DNA-protein complex formation by fluorescence anisotropy

Bahr, Matthias; Valis, Linda; Wagenknecht, Hans-Achim; Weinhold, Elmar, Nucleosides Nucleotides Nucleic Acids 2007, 26(10-12), 1581-1584.

In this work, fluorescence anisotropy was used to study DNA binding of the DNA methyltransferase M.TaqI. For this purpose short DNA molecules labelled with three different fluorophores (Cy3, thiazole orange, and ethidium bromide) were prepared in various topologies and their suitability for detection of DNA-protein complex formation was investigated.

2-Aminopurine Flipped into the Active Site of the Adenine-Specific DNA Methyltransferase M.TaqI: Crystal Structures and Time-Resolved Fluorescence

Lenz, Thomas; Bonnist, Eleanor Y. M.; Pljevaljcic, Goran; Neely, Robert K.; Dryden, David T. F.; Scheidig, Axel J.; Jones, Anita C.; Weinhold, Elmar, J. Am. Chem. Soc. 2007, 129(19), 6240-6248.

We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the cryst. complex faithfully reports this state: base flipping is accompanied by the loss of the very short (.apprx.50 ps) lifetime component assocd. with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by π-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In soln., the same distinctive fluorescence response confirms complete destacking from DNA and is also obsd. when the proposed key residue for base flipping by M.TaqI, the target base partner thymine, is substituted by an abasic site analog. The corresponding cocrystal structure shows 2-aminopurine in the active site of M.TaqI, demonstrating that the partner thymine is not essential for base flipping. However, in this structure, a shift of the 3' neighbor of the target base into the vacancy left after base flipping is obsd., apparently replicating a stabilizing role of the missing partner thymine. Time-resolved fluorescence and acrylamide quenching measurements of M.TaqI complexes in soln. provide evidence for an alternative binding site for the extra-helical target base within M.TaqI and suggest that the partner thymine assists in delivering the target base into the active site.

A new tool for biotechnology: AdoMet-dependent methyltransferases

Klimasauskas, Saulius; Weinhold, Elmar, Trends in Biotechnology 2007, 25(3), 99-104.

A review. S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases catalyze highly specific Me group transfers from the ubiquitous cofactor, S-adenosyl-L-methionine to a multitude of biol. targets in the cell. Recently, DNA methyltransferases have been used for the sequence-specific, covalent attachment of larger chem. groups to plasmid and bacteriophage DNA using 2 classes of synthetic AdoMet analogs. These synthetic cofactors, in combination with the myriad AdoMet-dependent methyltransferases available in nature, provide new mol. tools for precise, targeted functionalization and labeling of large natural DNAs and, in all likelihood, RNAs and proteins. This paves the way for numerous novel applications in the functional anal. of biol. methylation, biotechnol., and medical diagnostics.

Targeted Labeling of DNA by Methyltransferase-Directed Transfer of Activated Groups (mTAG)

Lukinavicius, Grazvydas; Lapiene, Vidmantas; Stasevskij, Zdislav; Dalhoff, Christian; Weinhold, Elmar; Klimasauskas, Saulius, J. Am. Chem. Soc. 2007, 129(10), 2758-2759.

Methyltransferases catalyze highly specific transfers of Me groups from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) to various biopolymers like DNA, RNA, and proteins. Here we describe the first synthetic analog of AdoMet with an activated side chain carrying a primary amino group that permits efficient methyltransferase-directed functionalization of DNA and subsequent amine-specific chemoligations with various reporter groups. The demonstrated two-step sequence-specific labeling of natural DNA offers a facile way to query the methylation status of the target sites and envisions numerous applications in functional studies and medical diagnostics.

Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases

Dalhoff, Christian; Lukinavicius, Grazvydas; Klimasauskas, Saulius; Weinhold, Elmar, Nature Chemical Biology 2006, 2(1), 31-32.

S-Adenosyl-L-methionine (AdoMet) is the major Me donor for biol. methylation reactions catalyzed by methyltransferases. We report the first chem. synthesis of AdoMet analogs with extended carbon chains replacing the Me group and their evaluation as cofactors for all three classes of DNA methyltransferases. Extended groups contg. a double or triple bond in the β position to the sulfonium center were transferred onto DNA in a catalytic and sequence-specific manner, demonstrating a high utility of such synthetic cofactors for targeted functionalization of biopolymers.

A fluorescent quinoline derivative as selective receptor for fluoride anions

Albrecht, Markus; Triyanti; de Groot, Marita; Bahr, Matthias; Weinhold, Elmar, Synlett 2005, 13, 2095-2097.

A fluorescent anion receptor based on a quinoline backbone and on amide and urea side chains is prepd. and fluorescence titrns. in chloroform reveal a high affinity for fluoride (Ka = 14400 M-1) over chloride (Ka = 3100 M-1) and bromide (Ka = 640 M-1).

Forced intercalation as a tool in gene diagnostics and in studying DNA-protein interactions

Koehler, Olaf; Jarikote, Dilip V.; Singh, Ishwar; Parmar, Virinder S.; Weinhold, Elmar; Seitz, Oliver, Pure and Applied Chemistry 2005, 77(1), 327-338.

Arom. and heteroarom. groups that are forced to intercalate at specific positions in DNA are versatile probes of DNA-DNA and DNA-protein recognition. Fluorescent nucleobases are of value since they are able to report on localized alterations of DNA duplex structure. However, the fluorescence of the vast majority of base surrogates becomes quenched upon intercalation in DNA. Peptide nucleic acid (PNA)-based probes are presented in which the intercalator dye thiazole orange (TO) serves as a fluorescent base surrogate. In these probes, fluorescence increases (5-60-fold) upon hybridization. PNA-bearing TO as fluorescent base surrogate could hence prove useful in real-time polymerase chain reaction (PCR) applications and in live cell anal. Forced intercalation of arom. polycycles can help to explore the binding mechanism of DNA-modifying enzymes. We discuss studies of DNA-methyltransferases (MTases) which commence methylation of nucleobases in DNA by flipping the target nucleotide completely out of the helix. A method for probing the base-flipping mechanism is suggested. It draws upon the observation that large hydrophobic base surrogates in the face of the swung-out base can enhance the DNA-enzyme binding affinity possibly by disrupting target base-stacking and stabilizing the apparent abasic site.

Abasic site stabilization by aromatic DNA base surrogates: High-affinity binding to a base-flipping DNA-methyltransferase

Singh, Ishwar; Beuck, Christine; Bhattacharya, Anupam; Hecker, Walburga; Parmar, Virinder S.; Weinhold, Elmar; Seitz, Oliver, Pure and Applied Chemistry 2004,76(7-8), 1563-1570.

DNA-methyltransferases catalyze the sequence-specific transfer of the Me group of S-adenosylmethionine to target bases in genomic DNA. For gaining access to their target embedded within a double-helical structure, DNA-methyltransferases (DNA-MTases) rotate the target base out of the DNA helix. This base-flipping leads to the formation of an apparent abasic site. MTases such as cytosine-specific M.HhaI and M.HaeIII and also the repair enzyme uracil DNA glycosylase (UDG) insert amino acid side chains into the opened space and/or rearrange base-pairing. The adenine-specific DNA MTase M.TaqI binds without amino acid insertion. This binding mode allows for a substitution of the orphaned thymine with larger DNA base surrogates without steric interference by inserted amino acid side chains. DNA contg. pyrenyl, naphthyl, acenaphthyl, and biphenyl residues was tested in M.TaqI binding studies. The synthesis of DNA building blocks required the formation of a C-glycosidic bond, which was established by using protected 1-chloro-2-deoxyribose as glycosyl donor and organocuprates as glycosyl acceptors. It is shown that all of the base surrogates enhanced the binding affinity to M.TaqI. Incorporation of pyrene increased the binding affinity by a factor of 400. Interestingly, there is a correlation between the obsd. order of dissocn. consts. and the ability of a base surrogate to stabilize abasic sites in model duplexes.

Sequence-specific DNA labeling using methyltransferases

Pljevaljcic, Goran; Schmidt, Falk; Peschlow, Alexander; Weinhold, Elmar, Methods in Molecular Biology (Totowa, NJ, United States) 2004, 283(Bioconjugation Protocols), 145-161.

Sequence-specific labeling of native DNA (DNA) still represents a more-or-less unsolved problem. Difficulties mainly arise from the necessity to combine two different functions: sequence-specific recognition of DNA and covalent bond formation between the label and DNA. DNA methyltransferases (MTases) naturally possess these two functions and transfer a Me group from the cofactor S-adenosyl-L-methionine (AdoMet) to adenine or cytosine residues within specific DNA sequences, typically ranging from two to eight base pairs. Unfortunately, the Me group itself is a very limited reporter group and it would be desirable to transfer larger chem. entities with DNA MTases. Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to the synthetic cofactor N-adenosylaziridine, which is quant., base- and sequence-specifically coupled with DNA in a DNA MTase-catalyzed reaction. By attaching interesting reporter groups to a suitable position of N-adenosylaziridine a large variety of new synthetic cofactors are obtained for sequence-specific labeling of DNA. This method is illustrated by coupling primary amino groups and biotin to short duplex oligodeoxynucleotides or plasmid DNA using the DNA MTase M.TaqI.

Sequence-specific methyltransferase-induced labeling of DNA (SMILing DNA)

Pljevaljcic, Goran; Schmidt, Falk; Weinhold, Elmar, ChemBioChem 2004, 5(3), 265-269.

A new concept for sequence-specific labeling of DNA by using chem. 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.Taql) 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 addn., this novel method for DNA labeling should be applicable to a wide variety of reporter groups.

Convenient Synthesis of Oligodeoxynucleotides Containing 2´-Deoxy-6-thioinosine

C. Beuck, E. Weinhold, Nucleosides, Nucleotides & Nucleic Acids 2003, 22(5-8), 635-639.

A facile synthesis of oligodeoxynucleotides (ODN) contg. 2´-deoxy-6-thioinosine (dI6S) based on the convertible nucleoside O6-phenyl-2´-deoxyinosine is presented. After std. solid-phase DNA synthesis and removal of the cyanoethyl protecting groups with DBU treatment with aq. sodium hydrogen sulfide introduces the sulfur functionality, deprotects the other nucleobases and cleaves the ODN from the solid support in a one-pot reaction. In addn., the extinction coeff. of 2´-deoxy-6-thioinosine is detd. by enzymic fragmentation of the resulting ODN in the presence of adenosine deaminase.

DNA mismatch-specific base flipping by a bisacridine macrocycle.

David, Arnaud; Bleimling, Nathalie; Beuck, Christine; Lehn, Jean-marie; Weinhold, Elmar; Teulade-fichou, Marie-paule, ChemBioChem 2003, 4(12), 1326-1331.

Most, if not all, enzymes that chem. modify nucleobases in DNA flip their target base from the inside of the double helix into an extra-helical position. This energetically unfavorable conformation is partly stabilized by specific binding of the apparent abasic site being formed. Thus, DNA base-flipping enzymes, like DNA methyltransferases and DNA glycosylases, generally bind very strongly to DNA contg. abasic sites or abasic-site analogs. The macrocyclic bisacridine BisA has previously been shown to bind abasic sites. Herein we demonstrate that it is able to specifically recognize DNA base mismatches and most likely induces base flipping. Specific binding of BisA to DNA mismatches was studied by thermal denaturation expts. by using short duplex oligodeoxynucleotides contg. central TT, TC, or TG mismatches or a TA match. In the presence of the macrocycle a strong increase in the melting temp. of up to 7.1° was obsd. for the mismatch-contg. duplexes, whereas the melting temp. of the fully matched duplex was unaffected. Furthermore, BisA binding induced an enhanced reactivity of the mispaired thymine residue in the DNA toward potassium permanganate oxidn. A comparable reactivity has previously been obsd. for a TT target base mismatch in the presence of DNA methyltransferase M•Taql. This similarity to a known base-flipping enzyme suggests that insertion of BisA into the DNA helix displaces the mispaired thymine residue into an extra-helical position, where it should be more prone to chem. oxidn. Thus, DNA base flipping does not appear to be limited to DNA-modifying enzymes but it is likely to also be induced by a small synthetic mol. binding to a thermodynamically weakened site in DNA.

Polycyclic aromatic DNA-base surrogates: High-affinity binding to an adenine-specific base-flipping DNA methyltransferase

C. Beuck, I. Singh, A. Bhattacharya, W. Hecker, V. S. Parmar, O. Seitz, E. Weinhold, Angewandte Chemie, International Edition 2003, 42(33), 3958-3960.

DNA methylation is catalyzed by DNA methyltransferases (MTases), which bind to specific DNA sequences and transfer a Me group from S-adenosyl-L-methionine to the exocyclic amino groups of adenine or cytosine or to C5 of cytosine. Interestingly, methylation is commenced by flipping the target nucleotide completely out of the helix. Different mechanisms of abasic-site stabilization have been obsd. in crystal structures of DNA MTases complexed with DNA. In the case of the adenine-specific DNA MTase M.TaqI the unpaired partner of the flipped base is inserted into the opened space, resulting in interstrand stacking. Inspection of the crystal structure of M.TaqI suggested that interstrand stacking could be improved by replacing the partner thymine with larger arom. DNA-base surrogates. It was expected that more complete filling of the apparent abasic site would lead to tightening of the M.TaqI-DNA complex. In addn., it was thought that a polycyclic base surrogate could destabilize the innerhelical conformation of the opposing target adenine. The DNA MTase could thus bind to double-stranded DNA contg. an unstacked target base without having to compensate for the energy required for disrupting Watson-Crick hydrogen bonds and base-stacking interactions. These hypotheses were investigated with DNA substrates that contained pyrenyl, 1-naphthyl, 2-naphthyl, acenaphthyl, and biphenyl residues opposite to the target position within the recognition sequence of M.TaqI.

The stability of pseudopeptides bearing sulfoximines as chiral backbone modifying element towards proteinase

C. Bolm, D. Müller, C. Dalhoff, C. P. R. Hackenberger, E. Weinhold, Bioorg. Med. Chem. Lett. 2003, 13(19), 3207-3211.

Syntheses of pseudopeptides (S)- or (R)-H-Phe-N:S(O)PhCH2CO-Phe-OH and (S)-HN:S(O)PhCH2CO-Phe-OH are described. Incorporation of sulfoximines as backbone modifying element results in two new pseudopeptide bonds which display enhanced (bond A) and strongly reduced reactivity (bond B) towards hydrolysis by proteinase K.

Design of a New Fluorescent Cofactor for DNA Methyltransferases and Sequence-Specific Labeling of DNA.

G. Pljevaljcic, M. Pignot, E. Weinhold, J. Am. Chem. Soc. 2003, 125(12), 3486-3492.

Sequence-specific labeling of DNA is of immense interest for anal. and functional studies of DNA. We present a novel approach for sequence-specific labeling of DNA using a newly designed fluorescent cofactor for the DNA methyltransferase from Thermus aquaticus (M.TaqI). Naturally, M.TaqI catalyzes the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded 5´-TCGA-3´ DNA sequence onto the Me group of the cofactor S-adenosyl-L-methionine (AdoMet) leading to Me group transfer. The design of a new fluorescent cofactor for covalent labeling of DNA was based on three criteria: (1) Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to M.TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to DNA. (2) The adenosyl moiety is the mol. anchor for cofactor binding. (3) Attachment of a fluorophore via a flexible linker to the 8-position of the adenosyl moiety does not block cofactor binding. According to these criteria the new fluorescent cofactor 8-amino[1´´-(N´´-dansyl)-4´´-aminobutyl]-5´-(1-aziridinyl)-5´-deoxyadenosine (3) was synthesized. 3 binds about 4-fold better than the natural cofactor AdoMet to M.TaqI and is coupled with a short duplex oligodeoxynucleotide by M.TaqI. The identity of the expected modified nucleoside was verified by electrospray ionization mass spectrometry after enzymic fragmentation of the product duplex. In addn., the new cofactor 3 was used to sequence-specifically label plasmid DNA in a M.TaqI-catalyzed reaction.

A DNA-binding peptide from a phage display library.

J. Wölcke, E. Weinhold, Nucleosides, Nucleotides & Nucleic Acids 2001, 20(4-7), 1239-1241.

A DNA-binding peptide was selected from a random peptide phage display library. For competitive elution using the DNA methyltransferase M . TaqI in the selection step, a biotin-labeled duplex oligodeoxyribonucleotide contg. the 5´-TCGA-3´ recognition sequence of M.TaqI was employed. Nine of ten phages selected were found to have the same deduced amino acid sequence SVSVGMKPSPRP. The selected phage binds to DNA, as demonstrated in an ELISA.

The mechanism of DNA cytosine-5 methylation. Kinetic and mutational dissection of HhaI methyltransferase.

G. Vilkaitis, E. Merkiene, S. Serva, E.Weinhold, S. Klimasauskas, J. Biol. Chem. 2001, 276(24), 20924-20934.

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex contg. a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-L-methionine (AdoMet) and product S-adenosyl-L-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in soln. The M.HhaI.AdoMet complex (koff = 22 s-1, KD = 6 mM) is partially converted into products during isotope-partitioning expts., suggesting that it is catalytically competent. Chem. formation of the product M.HhaI.MeDNA.AdoHcy (kchem = 0.26 s-1) is followed by a slower decay step (koff = 0.045 s-1), which is the rate-limiting step in the catalytic cycle (kcat = 0.04 s-1). Anal. of reaction products shows that the hemimethylated substrate undergoes complete (>95%) conversion into fully methylated product during the initial burst phase, indicating that M.HhaI exerts high binding selectivity toward the target strand. The T250N, T250D, and T250H mutations, which introduce moderate perturbation in the catalytic site, lead to substantially increased KDDNA(ternary), koffDNA(ternary), KMAdoMet(ternary) values but small changes in KDDNA(binary), KDAdoMet(binary), kchem, and kcat. When the target cytosine is replaced with 5-fluorocytosine, the chem. step leading to an irreversible covalent M.HhaI.DNA complex is inhibited 400-fold (kchem5FC = 0.7 * 10-3 s-1), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation kchem. We suggest that activation of the pyrimidine ring via covalent addn. at C-6 is a major contributor to the rate of the chem. step (kchem) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.

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

K. Goedecke, M. Pignot, R. S. Goody, A. J. Scheidig, E. Weinhold. Nature Struct. Biol. 2001, 8(2), 121-125.

The 2.0 .ANG. crystal structure of N6-adenine DNA methyltransferase TaqI in complex with specific DNA and a nonreactive cofactor analog, 5´-[2-(amino)ethylthio]-5´deoxyadenosine, revealed a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the Me group from the cofactor S-adenosyl-L-methionine to the 6-NH2 group of adenine within the double-stranded DNA sequence, 5´-TCGA-3´, the target nucleoside was rotated out of the DNA helix. Stabilization of the extrahelical conformation was 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 p-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine was specifically recognized in the active site, and the 6-NH2 group of adenine donated 2 H-bonds to Asn-105 and Pro-106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These H-bonds appeared to increase the partial neg. charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the Me group of the cofactor.

Functional roles of the conserved threonine 250 in the target recognition domain of HhaI DNA methyltransferase.

G. Vilkaitis, A. Dong, E. Weinhold, X. Cheng, S. Klimasauskas. J. Biol. Chem. 2000, 275(49), 38722-38730.

DNA cytosine-5-methyltransferase HhaI recognizes the GCGC sequence and flips the inner cytosine out of DNA helix and into the catalytic site for methylation. The 5´-phosphate of the flipped out cytosine is in contact with the conserved Thr-250 from the target recognition domain. We have produced 12 mutants of Thr-250 and examd. their methylation potential in vivo. Six active mutants were subjected to detailed biochem. and structural studies. Mutants with similar or smaller side chains (Ser, Cys, and Gly) are very similar to wild-type enzyme in terms of steady-state kinetic parameters kcat, KMDNA, KMAdoMet. In contrast, the mutants with bulkier side chains (Asn, Asp, and His) show increased KM values for both substrates. Fluorescence titrns. and stopped-flow kinetic anal. of interactions with duplex oligonucleotides contg. 2-aminopurine at the target base position indicate that the T250G mutation leads to a more polar but less solvent-accessible position of the flipped out target base. The x-ray structure of the ternary M.HhaI(T250G).DNA.AdoHcy complex shows that the target cytosine is locked in the catalytic center of enzyme. The space created by the mutation is filled by water mols. and the adjacent DNA backbone atoms dislocate slightly toward the missing side chain. In aggregate, our results suggest that the side chain of Thr-250 is involved in constraining the conformation the DNA backbone and the target base during its rotation into the catalytic site of enzyme.

Reviving a Dead Enzyme: Cytosine Deaminations Promoted by an Inactive DNA Methyltransferase and an S-Adenosylmethionine Analogue.

A. N. Sharath, E. Weinhold, A. S. Bhagwat. Biochemistry 2000, 39(47), 14611-14616.

The enzymes that transfer a Me group to C5 of cytosine within specific sequences (C5 Mtases) deaminate the target cytosine to uracil if the Me donor S-adenosylmethionine (SAM) is omitted from the reaction. Recently, it was shown that cytosine deamination caused by C5 Mtases M.HpaII, M.SssI and M.MspI is enhanced in the presence of several analogs of SAM, and a mechanism for this analog-promoted deamination was proposed. According to this mechanism, the analogs protonate C5 of the target cytosine, creating a dihydrocytosine intermediate that is susceptible to deamination. We show here that one of these analogs, 5´-aminoadenosine (AA), enhances cytosine deamination by the Mtase M.EcoRII, but it does so without enhancing protonation of C5. Further, we show that uracil is an intermediate in the mutational pathway and propose an alternate mechanism for the analog-promoted deamination. The new mechanism involves a facilitated water attack at C4 but does not require attack at C6 by the enzyme. The latter feature of the mechanism was tested by using M.EcoRII mutants defective in the nucleophilic attack at C6 in the deamination assay. We find that although these proteins are defective in Me transfer and cytosine deamination, they cause cytosine deaminations in the presence of AA in the reaction. Our results point to a possible connection between the catalytic mechanism of C5 Mtases and of enzymes that transfer Me groups to N4 of cytosine. Further, they provide an unusual example where a coenzyme activates an otherwise "dead" enzyme to perform catalysis by a new reaction pathway.

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

M. Pignot, G. Pljevaljcic, E. Weinhold, Eur. J. Org. Chem. 2000, 3, 549-555.

5´-Acetylthio-5´-deoxy-2´,3´-O-isopropylideneadenosine (I) was directly prepd. from com. available 2´,3´-O-isopropylideneadenosine and thioacetic acid under Mitsunobu conditions in almost quant. yield. In situ cleavage of the acetylthio function of I followed by coupling with different alkyl bromides proceeded with high yields. Deprotection of the obtained 5´-thionucleosides yielded the S-adenosyl-L-homocysteine analogs decarboxylated AdoHcy, deaminated AdoHcy and 5´-[3-(cyano)propylthio]-5´-deoxyadenosine in good overall yields. Direct deprotection of the thionucleoside I delivered 5´-thio-5´-deoxyadenosine in excellent yield. In addn., binding consts. of these AdoHcy analogs and the DNA methyltransferase M.HhaI were detd. in a fluorescence assay.

Probes for DNA base flipping by DNA methyltransferases.

B. Holz, E. Weinhold, (Hrsg.: U. Diederichsen, T. K. Lindhorst, B. Westermann, L. Wessjohann), in Bioorganic Chemistry: Highlights and New Aspects Wiley-VCH, Weinheim 1999, 337-345.

A review with 41 refs. is presented on probes for DNA base flipping by DNA methyltransferases. Specific topics discussed include (1) DNA methyltransferases and the biol. role of DNA methylation, (2) DNA base flipping as obsd. in X-ray structures of DNA methyltransferases, (3) 2-aminopurine in DNA as fluorescent probe for DNA base flipping, (4) tighter binding of modified DNA to DNA methyltransferases, (5) chem. detection of extrahelical bases in DNA-methyltransferase complexes and (6) other DNA-modifying enzymes using a DNA base flipping mechanism.

Aziridine-containing cofactors for methyltransferases and their use in labeling of nucleic acids and proteins.

M. Pignot, E. Weinhold, PCT-Patentanmeldung Nr. EP99/05405, Anmeldetag 28.07.1999.

Patent Family Information
Patent No. Kind Date Application No. Date
WO 2000006587 A1 20000210 WO 1999-EP5405 19990728
CA 2338721 AA 20000210 CA 1999-2338721 19990728
EP 1102781 A1 20010530 EP 1999-938363 19990728
JP 2002521488 T2 20020716 JP 2000-562384 19990728
Priority Application Information
EP    1998-114201   19980729
WO  1999-EP5405  19990728
Aziridine derivs. [I; X=N, CH; Y=N, CR3; R1,R3=H, 3H, NH(CH2)nNHR4, NH(C2H5O)nC2H5NHR4; R4=fluorophore, affinity tag, crosslinking agent, peptides, etc.; n=1-5000; R2=R1, CH2CH(COOH)(NH2)] are disclosed which can be used as cofactor for S-adenosyl-L-methionine-dependent methyltransferases. I and methyltransferases may be used to label nucleic acids and proteins. Thus, I (X,Y=N; R1,R2=H) was synthesized and used to label double-stranded oligonucleotide substrates of DNA methyltransferase TaqI and HhaI.

Higher binding affinity of duplex oligodeoxyribonucleotides containing 1,2-dideoxy-D-ribose to the N6-adenine DNA methyltransferase M.TaqI supports a base flipping mechanism.

B. Holz, E. Weinhold, Nucleosides & Nucleotides 1999,18,1355-1358.

A symposium on the N6-adenine DNA methyltransferase M.TaqI binds duplex oligodeoxyribonucleotides contg. 1,2-dideoxy-D-ribose at the target or 3´ neighboring position with 200 fold and 60 fold higher affinity, resp., compared to the natural substrate. These results are explained thermodynamically by a base flipping mechanism of M.TaqI.

Identification of the binding site for the extrahelical target base in N6-adenine DNA methyltransferases by photo-cross-linking with duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position.

B. Holz, N. Dank, J. E. Eickhoff, G. Lipps, G. Krauss, E. Weinhold, J. Biol. Chem. 1999, 274, 15066-15072.

DNA methyltransferases flip their target bases out of the DNA double helix for catalysis. Base flipping of C5-cytosine DNA methyltransferases was directly obsd. in the protein-DNA cocrystal structures of M.HhaI and M.HaeIII. Indirect structural evidence for base flipping of N6-adenine and N4-cytosine DNA methyltransferases was obtained by modeling DNA into the three-dimensional structures of M.TaqI and M.PvuII in complex with the cofactor. In addn., biochem. evidence of base flipping was reported for different N6-adenine DNA methyltransferases. An no protein-DNA cocrystal structure for the related N6-adenine and N4-cytosine DNA methyltransferases is available, we used light-induced photochem. crosslinking to identify the binding site of the extrahelical target bases. The N6-adenine DNA methyltransferases M.TaqI and M.CviBIII, which both methylate adenine within the double-stranded 5´-TCGA-3´ DNA sequence, were photo-cross-linked to duplex oligodeoxyribonucleotides contg. 5-iodouracil at the target position in 50-60% and almost quant. yield, resp. Proteolytic fragmentation of the M.CviBIII-DNA complex followed by Edman degrdn. and electrospray ionization mass spectrometry indicates photo-crosslinking to tyrosine 122. In addn., the mutant methyltransferases M.TaqI/Y108A and M.TaqI/F196A were photo-cross-linked with 6-fold and 2-fold reduced efficiency, resp., which suggests that tyrosine 108 is the primary site of modification in M.TaqI. Our results indicate a close proximity between the extrahelical target base and tyrosine 122 in M.CviBIII or tyrosine 108 in M.TaqI. As both residues belong to the conserved motif IV ((N/D/S)(P/I)P(Y/F/W)) found in all N6-adenine and N4-cytosine DNA as well as in N6-adenine RNA methyltransferases, a similar spatial relationship between the target bases and the arom. amino acid residue within motif IV is expected for all these methyltransferases.

Functional roles of the conserved aromatic amino acid residues at position 108 (motif IV) and position 196 (motif VIII) in base flipping and catalysis by the N6-adenine DNA methyltransferase from Thermus aquaticus.

H. Pues, N. Bleimling, B. Holz, J. Wölcke, E. Weinhold, Biochemistry 1999, 38, 1426-1434.

DNA methyltransferase TaqI (I) from T. aquaticus catalyzes the transfer of the activated Me group of S-adenosyl-L-methionine to the N6 position of adenine within double-stranded DNA sequence 5´-TCGA-3´. To achieve catalysis I flips the target adenine out of the DNA helix. On the basis of the 3-dimensional structure of I in complex with the cofactor and its structural homol. to the C5-cytosine DNA methyltransferase HhaI from Haemophilus haemolyticus, Tyr-108 and Phe-196 were suggested to interact with the extrahelical adenine. The functional roles of these 2 arom. amino acid residues in I were investigated by mutational anal. The obtained mutant I were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide contg. the fluorescent base analog, 2-aminopurine, at the target position. Whereas the mutant I contg. the arom. amino acid, Trp, at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant I with the nonarom. amino acid, Ala (Y108A and F196A), had a strongly reduced kcat. However, mutant Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicated that Phe-196 is important for stabilizing the extrahelical target adenine and suggest that Tyr-108 is involved in placing the extrahelical target base in an optimal position for Me group transfer. Since both arom. amino acids belong to conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA methyltransferases as well as in N6-adenine RNA methyltransferases, a similar function of arom. amino acid residues within these motifs is expected for the different methyltransferases.

Coupling of a nucleoside with DNA by a methyltransferase.

M. Pignot, C. Siethoff, M. Linscheid, E. Weinhold, Angew. Chem.1998, 110, 3050-3053; Angew. Chem. Int. Ed. 1998, 37, 2888-2891.

S-Adenosyl-L-methionine-dependent methyltransferase (Mtases) catalyze the transfer of the activated Me group from the cofactor S-adenosyl-L-methionine to sulfur, nitrogen, oxygen and carbon acceptors of small mols., phospholipids, RNA and DNA with specificity. The authors present the first example of a Mtase-catalyzed formation of a covalent bond between a group larger than a Me group and the substrate for a Mtase. N-adenosylaziridine was synthesized and tested as a substrate for Thermus aquaticus DNA Mtase.

Chemical display of thymine residues flipped out by DNA methyltransferases.

S. Serva, E. Weinhold, R. J. Roberts, S. Klimasauskas, Nucleic Acids Res. 1998, 26, 3473-3479.

The DNA cytosine-C5 methyltransferase M.HhaI flips its target base out of the DNA helix during interaction with the substrate sequence GCGC. Binary and ternary complexes between M.HhaI and hemimethylated DNA duplexes were used to examine the suitability of four chem. methods to detect flipped-out bases in protein-DNA complexes. These methods probe the structural peculiarities of pyrimidine bases in DNA. We find that in cases when the target cytosine is replaced with thymine (GTGC), KMnO4 proved an efficient probe for pos. display of flipped-out thymines. The generality of this procedure was further verified by examg. a DNA adenine-N6 methyltransferase, M.TaqI, in which case an enhanced reactivity of thymine replacing the target adenine (TCGT) in the recognition sequence TCGA was also obsd. Our results support the proposed base-flipping mechanism for adenine methyltransferases, and offer a convenient lab. tool for detection of flipped-out thymines in protein-DNA complexes.

2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases.

B. Holz, S. Klimasauskas, S. Serva, E. Weinhold, Nucleic Acids Res. 1998, 26, 1076-1083.

DNA base flipping, which was first obsd. for the C5-cytosine DNA methyltransferase M.HhaI, results in a complete removal of the stacking interactions between the target base and its neighboring bases. We have investigated whether duplex oligodeoxynucleotides contg. the fluorescent base analog 2-aminopurine can be used to sense DNA base flipping. Using M.HhaI as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides contg. 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M.HhaI. Duplex oligodeoxynucleotides contg. 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addn. of M.HhaI. These results clearly demonstrate that duplex oligodeoxynucleotides contg. 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M.TaqI. Addn. of M.TaqI to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addn. to duplex oligodeoxynucleotides contg. 2-aminopurine at the 3´- or 5´-neighboring position leads only to small fluorescence increases. These results give the first exptl. evidence that the adenine-specific DNA methyltransferase M.TaqI also flips its target base.

Construction of a deletion library using a mixture of 5´-truncated primers for inverse PCR (IPCR).

H. Pues, B. Holz, E. Weinhold, Nucleic Acids Res. 1997, 25, 1303-1304.

A quick in vitro mutagenesis method for the construction of nested deletion libraries was developed. Many deletions can be obtained in a single inverse PCR (IPCR) by replacing one of the two primers with a mixt. of 5´-truncated oligodeoxynucleotides. Since chem. DNA synthesis proceeds from the 3´ to the 5´ end, such a mixt. of 5´-truncated oligodeoxynucleotides can easily be obtained in a single automated DNA synthesis under reduced coupling efficiency. This deletion mutagenesis method yields many different deletions in a defined short DNA segment and is, therefore, best suited for a deletion anal. at base pair level. Applications might include functional anal. of regulatory DNA segments and protein engineering work that requires libraries for the expression of N-terminal, C-terminal or internal truncated proteins as well as fusion proteins having different splice sites.

Differential binding of S-adenosylmethionine S-adenosylhomocysteine and sinefungin to the adenine-specific DNA methyltransferase M. TaqI.

G. Schluckebier, M. Kozak, N. Bleimling, E. Weinhold, W. Saenger, J. Mol. Biol. 1997, 265, 56-67.

The crystal structures of the binary complexes of DNA modification methyltransferase TaqI (I) with the inhibitor, sinefungin (II), and the reaction product, S-adenosyl-L-homocysteine (III), were detd., both at 2.6 .ANG. resoln. Structural comparison of these binary complexes with the complex formed by I and the cofactor, S-adenosyl-L-methionine (IV), suggested that the key element for mol. recognition of these ligands is the binding of the adenosine moiety in a pocket, and discrimination between cofactor, reaction product, and inhibitor is mediated by the different conformations of these mols.; the methionine moiety of IV was located in the binding cleft, whereas the amino acid moieties of II and III were in a different orientation and interacted with the active site amino acid residues, 105NPPY108. The KD values for I complexes with the 3 ligands were detd. spectrofluorometrically. II bound more strongly than III or IV, with KD values of 0.34, 2.4, and 2.0 mM, resp.

Fluorescence studies of the DNA base flipping induced by a cytosine-5 methyltransferase.

S. Serva, S. Klimasauskas, E. Weinhold, Biologija 1997, 9-12.

A novel fluorescence-based method for detecting and studying DNA base flipping in enzyme-DNA complexes is described. The target cytosine for the DNA methyltransferase HhaI (I) recognition sequence (GCGC) was replaced by a fluorescent base, 2-aminopurine [G(2AP)GC]. Consistent with the extrahelical trapping of the target base, a 90-fold increase in the fluorescence intensity was obsd. upon binding of I to a 37-mer duplex substrate. Similar substitutions of bases adjacent to the target cytosine resulted in a relatively small change. Stopped-flow fluorescence expts. under noncatalytic conditions allowed one to monitor the course of base-flipping directly. Kinetic anal. showed that the site-specific binding of the enzyme was a diffusion-controlled process, whereas the subsequent flipping motion was achieved in 1 ms or faster. Other classes of enzymes suspected to employ base flipping in their mechanisms can be investigated with this method.

Engineering yeast alcohol dehydrogenase. Replacing Trp54 by Leu broadens substrate specificity.

E. G. Weinhold, S. A. Benner, Protein Engineering 1995, 8, 457-461.

Anal. of the crystal structure of alc. dehydrogenase (Adh) from horse liver suggests that Trp-54 in the homologous yeast alc. dehydrogenase prevents the yeast enzyme from efficiently catalyzing the oxidn. of long-chain primary alcs. with branching at the 4 position (e.g., 4-methyl-2-pentanol, cinnamyl alc.). Here, this residue was altered to Leu by site-directed mutagenesis (W54L mutant). The alteration yielded an enzyme that served as an effective catalyst for both longer straight-chain primary alcs. and branched chain alcs.

Design and evaluation of a tightly binding fluorescent ligand for influenza A hemagglutinin.

E. G. Weinhold, J. R. Knowles, J. Am. Chem. Soc. 1992, 114, 9270-9275.

Attachment of influenza virus to susceptible cells is mediated by the viral protein hemagglutinin, which recognizes cell-membrane-bound glycoconjugates that terminate in a-sialosides. Fluorescent a-sialosides, I (R = OMe, R1) were prepd. in 6 steps. Sialoside I (R = R1) has the highest affinity of any reported monovalent ligand for hemagglutinin and provides a convenient fluorescence competition assay for the binding of other ligands, and it is not a substrate for the viral neuraminidase. Since each of the currently used binding assays has significant disadvantages, such a simple assay is of great importance for the study of potential inhibitors of viral attachment.

Mechanistic and active-site studies on D(-)-mandelate dehydrogenase from Rhodotorula graminis.

D. P. Baker, C. Kleanthous, J. N. Keen, E. Weinhold, C. A. Fewson, Biochem. J. 1992, 281, 211-218.

D(-)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast R. graminis, catalyzes the NAD-dependent oxidn. of D(-)-mandelate to phenylglyoxylate. D(-)-2-(Bromoethanoyloxy)-2-phenylethanoic acid [D(-)-bromoacetylmandelic acid], an analog of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(-)-Mandelate dehydrogenase was inactivated by D(-)-bromoacetylmandelate in a pseudo-first-order process. D(-)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(-)-Mandelate dehydrogenase that had been inactivated with 14C-labeled D(-)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into 2 tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the unidentified residue in the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not detd., but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N´-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(-)-bromoacetylmandelate reacts. D(-)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(-)-mandelate and NAD were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the redn. of phenylglyoxylate. The results are discussed with particular ref. to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.

Structural determinants of stereospecificity in yeast alcohol dehydrogenase.

E. G. Weinhold, A. Glasfeld, A. D. Ellington, S. A. Benner, Proc. Nat. Acad. Sci. USA 1991, 88, 8420-8424.

Replacing Leu-182 by Ala in yeast alc. dehydrogenase (YADH; alc.:NAD+ oxidoreductase, EC yields a mutant that retains 34% of its kcat value and makes one stereochem. mistake every 850,000 turnovers (instead of 1 error every 7,000,000,000 turnovers in native YADH) in its selection of the 4-Re hydrogen of NADH. Half of the decrease in stereochem. fidelity comes from an increase in the rate of transfer of the 4-Si hydrogen of NADH. The mutant also accepts 5-methylnicotinamide adenine dinucleotide, a cofactor analog not accepted by native YADH. The stereospecificity of the mutant is lower still with analogs of NADH where the carboxamide group of the nicotinamide ring is replaced by groups with weaker hydrogen bonding potential. For example, with thio-NADH, the mutant enzyme makes 1 stereochem. mistake every 450 turnovers. Finally, the double mutant T157S/L182A, in which Thr-157 is replaced by Ser and Leu-182 is replaced by Ala, also shows decreased stereochem. fidelity. These results suggest that Si transfer in the mutant enzymes arises from NADH bound in a syn conformation in the active site and that this binding is not obstructed in native YADH by side chains essential for catalysis.

Electrophilic additions to 3-C-[(methoxycarbonyl)methyl]-3-deoxy-D-ribofuranose enolates: a case of unusually efficient nonchelate-enforced chirality transfer.

J. Mulzer, U. Steffen, L. Zorn, C. Schneider, E. Weinhold, W. Münch, R. Rudert, P. Luger, H. Hartl, J. Am. Chem. Soc. 1988, 110, 4640-4646.

The enolates I (R1 = Li, R2 = Me; R1 = Me, R2 = Li) obtained from ester II (R12 = Me2C, R2 = Me, R3 = R4 = H) on deprotonation with lithium diisopropylamide in THF and THF/HMPA, resp., add alkyl halides and benzaldehyde with >99% diastereoselectivity to form II [R12 = Me2C, R2 = Me, R3 = Me, CH2:CHCH2, PhCH2, CH(OH)Ph, R4 = H]. The structure of II (R12 = Me2C, R2 = Me3C, R3 = R4 = H) was established by x-ray crystallog. via the corresponding 5,6-diol. This unusually high chirality transfer is not due to a chelate-controlled mechanism, as the partially deoxygenated derivs. III (R1 = Bu, R2 = H, R3 = Me; R1 = Bu, R2 = R3 = Me; R1 = PhCH2CH2, R2 = H, R3 = Me3C) show the same or a slightly diminished selectivity on deprotonation/methylation. The steric course of the enolate alkylation may be described as a frontside attack of the electrophile on reactive conformation A. Furthermore, the stereochem. of the deprotonation of II (R12 = Me2C, R2 = Me, Me3C, R3 = R4 = H) was investigated by using the stereospecifically deuterated model compds. It was shown that the amide base attacks from the front side of the mol., the reactive conformation of the substrate now being of type B, in contrast to the alkylation.
Original design by: Christian Dalhoff
Last modified : 19.03.2013 (B.Jung)