Exposition of a family of RNA m(5)C methyltransferases from searching genomic and proteomic sequences.

The Escherichia coli fmu gene product has recently been determined to be the 16S rRNA m(5)C 967 methyltransferase. As such, Fmu represents the first protein identified as an S -adenosyl-L-methionine (AdoMet)- dependent RNA m(5)C methyltransferase whose amino acid sequence is known. Using the amino acid sequence of Fmu as an initial probe in an iterative search of completed DNA sequence databases, 27 homologous ORF products were identified as probable RNA m(5)C methyltransferases. Further analysis of sequences in undeposited genomic sequencing data and EST databases yielded more than 30 additional homologs. These putative RNA m(5)C methyltransferases are grouped into eight subfamilies, some of which are predicted to consist of direct genetic counterparts, or orthologs. The enzymes proposed to be RNA m(5)C methyltransferases have sequence motifs closely related to signature sequences found in the well-studied DNA m(5)C methyltransferases and other AdoMet-dependent methyltransferases. Structure-function correlates in the known AdoMet methyltransferases support the assignment of this family as RNA m(5)C methyltransferases.     

An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases

Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase.     

Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases

S-Adenosyl-L-methionine (AdoMet) is the major methyl donor for biological methylation reactions catalyzed by methyltransferases. We report the first chemical synthesis of AdoMet analogs with extended carbon chains replacing the methyl group and their evaluation as cofactors for all three classes of DNA methyltransferases. Extended groups containing a double or triple bond in the beta 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.     

Synthesis of TDP-3-amino-3,4,6-trideoxy-alpha-D-xylo-hexopyranose--the immediate precursor of TDP-alpha-D-desosamine

A synthetic pathway producing the title compound starting from methyl alpha-D-glucose is described. This compound was shown to be a substrate for DesVI, an AdoMet-dependent methyltransferase which catalyzes N,N-dimethylation of the title compound to give a biological significant unusual sugar, desosamine.     

Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase

AdoMet-dependent methylation of histones is part of the "histone code" that can profoundly influence gene expression. We describe the crystal structure of Neurospora DIM-5, a histone H3 lysine 9 methyltranferase (HKMT), determined at 1.98 A resolution, as well as results of biochemical characterization and site-directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for methyl transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the HKMT family and the SET domain.     

Simultaneous DNA binding, bending, and base flipping: evidence for a novel M.EcoRI methyltransferase-DNA complex

We measured the kinetics of DNA bending by M.EcoRI using DNA labeled at both 5'-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3'-ends, the effect is cofactor- and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from M.EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex. The M.EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly, M.EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes. Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.     

S-adenosyl-L-methionine-dependent methyl transfer: observable precatalytic intermediates during DNA cytosine methylation

S-adenosyl-L-methionine- (AdoMet-) dependent methyltransferases are widespread, play critical roles in diverse biological pathways, and are antibiotic and cancer drug targets. Presently missing from our understanding of any AdoMet-dependent methyl-transfer reaction is a high-resolution structure of a precatalytic enzyme/AdoMet/DNA complex. The catalytic mechanism of DNA cytosine methylation was studied by structurally and functionally characterizing several active site mutants of the bacterial enzyme M.HhaI. The 2.64 A resolution protein/DNA/AdoMet structure of the inactive C81A M.HhaI mutant suggests that active site water, an approximately 13 degree tilt of the target base toward the active site nucleophile, and the presence or absence of the cofactor methylsulfonium are coupled via a hydrogen-bonding network involving Tyr167. The active site in the mutant complex is assembled to optimally align the pyrimidine for nucleophilic attack and subsequent methyl transfer, consistent with previous molecular dynamics ab initio and quantum mechanics/molecular mechanics calculations. The mutant/DNA/AdoHcy structure (2.88 A resolution) provides a direct comparison to the postcatalytic complex. A third C81A ternary structure (2.22 A resolution) reveals hydrolysis of AdoMet to adenosine in the active site, further validating the coupling between the methionine portion of AdoMet and ultimately validating the structural observation of a prechemistry/postchemistry water network. Disruption of this hydrogen-bonding network by a Tyr167 to Phe167 mutation does not alter the kinetics of nucleophilic attack or methyl transfer. However, the Y167F mutant shows detectable changes in kcat, caused by the perturbed kinetics of AdoHcy release. These results provide a basis for including an extensive hydrogen-bonding network in controlling the rate-limiting product release steps during cytosine methylation.     

Automated identification of putative methyltransferases from genomic open reading frames

We have analyzed existing methodologies and created novel methodologies for the automatic assignment of S-adenosylmethionine (AdoMet)-dependent methyltransferase functionality to genomic open reading frames based on predicted protein sequences. A large class of the AdoMet-dependent methyltransferases shares a common binding motif for the AdoMet cofactor in the form of a seven-strand twisted beta-sheet; this structural similarity is mirrored in a degenerate sequence similarity that we refer to as methyltransferase signature motifs. These motifs are the basis of our assignments. We find that simple pattern matching based on the motif sequence is of limited utility and that a new method of "sensitized matrices for scoring methyltransferases" (SM2) produced with modified versions of the MEME and MAST tools gives greatly improved results for the Saccharomyces cerevisiae yeast genome. From our analysis, we conclude that this class of methyltransferases makes up approximately 0.6-1.6% of the genes in the yeast, human, mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Escherichia coli genomes. We provide lists of unidentified genes that we consider to have a high probability of being methyltransferases for future biochemical analyses.     

A conformational switch in the active site of BT_2972, a methyltransferase from an antibiotic resistant pathogen B. thetaiotaomicron

Methylation is one of the most common biochemical reactions involved in cellular and metabolic functions and is catalysed by the action of methyltransferases. Bacteroides thetaiotaomicron is an antibiotic-resistant bacterium that confers resistance through methylation, and as yet, there is no report on the structure of methyltransferases from this bacterium. Here, we report the crystal structure of an AdoMet-dependent methyltransferase, BT_2972 and its complex with AdoMet and AdoHcy for B. thetaiotaomicron VPI-5482 strain along with isothermal titration calorimetric assessment of the binding affinities. Comparison of the apo and complexed BT_2972 structures reveals a significant conformational change between open and closed forms of the active site that presumably regulates the association with cofactors and may aid interaction with substrate. Together, our analysis suggests that BT_2972 is a small molecule methyltransferase and might catalyze two O-methylation reaction steps involved in the ubiquinone biosynthesis pathway.     

Studies on naturally occurring proteinous inhibitor for transmethylation reactions

An inhibitor for S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two-dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyltransferases examined.     

Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase

We employed a highly specific photoaffinity labeling procedure, using (14)C-labeled S-adenosyl-l-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-(14)C]AdoMet or [carboxyl-(14)C]AdoMet yielded the sequence H(2)N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu(10654) and Pro(10655) as the major sites of derivatization. [carboxyl-(14)C]AdoMet in addition labeled Tyr(10650). Chymotryptic digestion generated the radiolabeled peptide H(2)N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu(2128) and Pro(2129), which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.     

S-adenosyl-L-homocysteine hydrolase and methylation disorders: Yeast as a model system

S-adenosyl-L-methionine (AdoMet)-dependent methylation is central to the regulation of many biological processes: more than 50 AdoMet-dependent methyltransferases methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids. Common to all AdoMet-dependent methyltransferase reactions is the release of the strong product inhibitor S-adenosyl-L-homocysteine (AdoHcy), as a by-product of the reaction. S-adenosyl-L-homocysteine hydrolase is the only eukaryotic enzyme capable of reversible AdoHcy hydrolysis to adenosine and homocysteine and, thus, relief from AdoHcy inhibition. Impaired S-adenosyl-L-homocysteine hydrolase activity in humans results in AdoHcy accumulation and severe pathological consequences. Hyperhomocysteinemia, which is characterized by elevated levels of homocysteine in blood, also exhibits a similar phenotype of AdoHcy accumulation due to the reversal of the direction of the S-adenosyl-L-homocysteine hydrolase reaction. Inhibition of S-adenosyl-L-homocysteine hydrolase is also linked to antiviral effects. In this review the advantages of yeast as an experimental system to understand pathologies associated with AdoHcy accumulation will be discussed.     

Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm,Bombyx mori

DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn²⁺ and Mn²⁺. Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1.     

AdoMet-dependent methylation, DNA methyltransferases and base flipping

Twenty AdoMet-dependent methyltransferases (MTases) have been characterized structurally by X-ray crystallography and NMR. These include seven DNA MTases, five RNA MTases, four protein MTases and four small molecule MTases acting on the carbon, oxygen or nitrogen atoms of their substrates. The MTases share a common core structure of a mixed seven-stranded beta-sheet (6 downward arrow 7 upward arrow 5 downward arrow 4 downward arrow 1 downward arrow 2 downward arrow 3 downward arrow) referred to as an 'AdoMet-dependent MTase fold', with the exception of a protein arginine MTase which contains a compact consensus fold lacking the antiparallel hairpin strands (6 downward arrow 7 upward arrow). The consensus fold is useful to identify hypothetical MTases during structural proteomics efforts on unannotated proteins. The same core structure works for very different classes of MTase including those that act on substrates differing in size from small molecules (catechol or glycine) to macromolecules (DNA, RNA and protein). DNA MTases use a 'base flipping' mechanism to deliver a specific base within a DNA molecule into a typically concave catalytic pocket. Base flipping involves rotation of backbone bonds in double-stranded DNA to expose an out-of-stack nucleotide, which can then be a substrate for an enzyme-catalyzed chemical reaction. The phenomenon is fully established for DNA MTases and for DNA base excision repair enzymes, and is likely to prove general for enzymes that require access to unpaired, mismatched or damaged nucleotides within base-paired regions in DNA and RNA. Several newly discovered MTase families in eukaryotes (DNA 5mC MTases and protein arginine and lysine MTases) offer new challenges in the MTase field.     

Mammalian DNA methyltransferases prefer poly(dI-dC) as substrate

The synthetic duplex DNA, poly(dI-dC).poly(dI-dC), is methylated in vitro by human or murine DNA methyltransferases at 20-100 times the rate of other nonmethylated DNAs. Preparation of the hemimethylated derivative, poly(dI-dMeC).poly(dI-dC), of this polymer increases its effectiveness as a substrate by 2-fold, making it 4-10 times more effective as a substrate for mammalian DNA methyltransferases than any other hemimethylated DNA so far reported. However, the apparent slower rate of de novo methylation of poly(dI-dC).poly(dI-dC) as compared to the hemimethylated derivative is due to substrate inhibition, unique to the unmethylated polymer, as the rates of de novo and maintenance methylation are identical at low substrate concentrations.     

Avidin plate assay system for enzymatic characterization of a histone lysine methyltransferase

Modification of proteins by protein methyltransferases has several important biological functions. Here, we study the methylation of histone H3 tail at position Lys9 by the Dim-5 histone lysine methyltransferase, which is involved in epigenetic signaling and gene silencing and which triggers DNA methylation in Neurospora crassa. We have developed a new assay to detect protein methylation using a biotinylated synthetic peptide substrate and a radioactively labeled coenzyme. We show that the assay is linear with respect to time and enzyme concentration (under multiple turnover conditions) and that its background is very low. Data points were reproducible within 3%. At least 200 pmol of biotinylated peptide is bound completely to the microplate. We employed the assay system to determine the K(m) and k(cat) values of the Dim-5 enzyme for the methylation of a 20 mer peptide to be 7.4 microM and 2.3 min(-1), respectively. In addition, we determined the activity of four Dim-5 variants, ranging from full activity to less than 1% of residual activity. The microplate biotin/avidin peptide methylation assay developed here is convenient, very accurate, reproducible, and inexpensive. Because it yields quantitative results, it can be employed for a characterization of the enzymatic properties of histone lysine methyltransferases and other protein methyltransferases. The assay also is well suited for high-throughput applications.