Sinefungin Research Articles

Effect of Withaferin-A, Withanone, and Caffeic Acid Phenethyl Ester on DNA Methyltransferases: Potential in Epigenetic Cancer Therapy.

DNA methyltransferases (DNMTs) have been reported to be potential drug targets in various cancers. The major hurdle in inhibiting DNMTs is the lack of knowledge about different DNMTs and their role in the hypermethylation of gene promoters in cancer cells. Lack of information on specificity, stability, and higher toxicity of previously reported DNMT inhibitors is the major reason for inadequate epigenetic cancer therapy. DNMT1 and DNMT3A are the two DNMTs that are majorly overexpressed in cancers. In this study, we have presented computational and experimental analyses of the potential of some natural compounds, withaferin A (Wi-A), withanone (Wi-N), and caffeic acid phenethyl ester (CAPE), as DNMT inhibitors, in comparison to sinefungin (SFG), a known dual inhibitor of DNMT1 and DNMT3A. We used classical simulation methods, such as molecular docking and molecular dynamics simulations, to investigate the binding potential and properties of the test compounds with DNMT1 and DNMT3A. Cell culture-based assays were used to investigate the inactivation of DNMTs and the resulting hypomethylation of the p16INK4A promoter, a key tumour suppressor that is inactivated by hypermethylation in cancer cells, resulting in upregulation of its expression. Among the three test compounds (Wi-A, Wi-N, and CAPE), Wi-A showed the highest binding affinity to both DNMT1 and DNMT3A; CAPE showed the highest affinity to DNMT3A, and Wi-N showed a moderate affinity interaction with both. The binding energies of Wi-A and CAPE were further compared with SFG. Expression analysis of DNMTs showed no difference between control and treated cells. Cell viability and p16INK4A expression analysis showed a dose-dependent decrease in viability, an increase in p16INK4A, and a stronger effect of Wi-A compared to Wi-N and CAPE. The study demonstrated the differential binding ability of Wi-A, Wi-N, and CAPE to DNMT1 and DNMT3A, which was associated with their inactivation, leading to hypomethylation and desilencing of the p16INK4A tumour suppressor in cancer cells. The test compounds, particularly Wi-A, have the potential for cancer therapy.

Investigating the effects of four medicinal plants against dengue virus through QSAR modeling and molecular dynamics studies

The Dengue virus (DENV) has been increasingly recognized as a prevalent viral pathogen responsible for global transmission of infection. It has been established that DENV's NS5 methyltransferase (MTase) controls viral replication. As a result, NS5 MTase is considered a potentially useful drug target for DENV. In this study, the two phases of virtual screening were conducted using the ML-based QSAR model and molecular docking to identify potential compounds against NS5 of DENV. Four medicinal plants [Aloe vera, Cannabis sativa (Hemp), Ocimum sanctum (Holy Basil; Tulsi), and Zingiber officinale (Ginger)] that showed anti-viral properties were selected for sourcing the phytochemicals and screening them against NS5. Additionally, re-docking at higher exhaustiveness and interaction analysis were performed which resulted in the identification of the top four hits (135398658, 5281675, 119394, and 969516) which showed comparable results with the control Sinefungin (SFG). Post molecular dynamics simulation, 135398658 showed the lowest RMSD (0.4–0.5 nm) and the maximum number of hydrogen bonds (eight hydrogen bonds) after the control while 5281675 and 969516 showed comparable hydrogen bonds to the control. These compounds showed direct interactions with the catalytic site residues GLU111 and ASP131, in addition to this these compounds showed stable complex formation as depicted by principal component analysis and free energy landscape. 135398658 showed lower total binding free energy (ΔG Total = −36.56 kcal/mol) than the control, while 5281675 had comparable values to the control (ΔG Total = −34.1 kcal/mol). Overall, the purpose of this study was to identify phytochemicals that inhibit NS5 function, that could be further tested experimentally to treat dengue virus (DENV). Communicated by Ramaswamy H. Sarma

Computational Analysis of SAM Analogs as Methyltransferase Inhibitors of nsp16/nsp10 Complex from SARS-CoV-2.

Methyltransferases (MTases) enzymes, responsible for RNA capping into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are emerging important targets for the design of new anti-SARS-CoV-2 agents. Here, analogs of S-adenosylmethionine (SAM), obtained from the bioisosteric substitution of the sulfonium and amino acid groups, were evaluated by rigorous computational modeling techniques such as molecular dynamics (MD) simulations followed by relative binding free analysis against nsp16/nsp10 complex from SARS-CoV-2. The most potent inhibitor (2a) shows the lowest binding free energy (-58.75 Kcal/mol) and more potency than Sinefungin (SFG) (-39.8 Kcal/mol), a pan-MTase inhibitor, which agrees with experimental observations. Besides, our results suggest that the total binding free energy of each evaluated SAM analog is driven by van der Waals interactions which can explain their poor cell permeability, as observed in experimental essays. Overall, we provide a structural and energetic analysis for the inhibition of the nsp16/nsp10 complex involving the evaluated SAM analogs as potential inhibitors.

Discovery of Inhibitors of DNA Methyltransferase 2, an Epitranscriptomic Modulator and Potential Target for Cancer Treatment.

Selective manipulation of the epitranscriptome could be beneficial for the treatment of cancer and also broaden the understanding of epigenetic inheritance. Inhibitors of the tRNA methyltransferase DNMT2, the enzyme catalyzing the S-adenosylmethionine-dependent methylation of cytidine 38 to 5-methylcytidine, were designed, synthesized, and analyzed for their enzyme-binding and -inhibiting properties. For rapid screening of potential DNMT2 binders, a microscale thermophoresis assay was established. Besides the natural inhibitors S-adenosyl-l-homocysteine (SAH) and sinefungin (SFG), we identified new synthetic inhibitors based on the structure of N-adenosyl-2,4-diaminobutyric acid (Dab). Structure-activity relationship studies revealed the amino acid side chain and a Y-shaped substitution pattern at the 4-position of Dab as crucial for DNMT2 inhibition. The most potent inhibitors are alkyne-substituted derivatives, exhibiting similar binding and inhibitory potencies as the natural compounds SAH and SFG. CaCo-2 assays revealed that poor membrane permeabilities of the acids and rapid hydrolysis of an ethylester prodrug might be the reasons for the insufficient activity in cellulo.

Virtual screening of molecular databases for potential inhibitors of the NSP16/NSP10 methyltransferase from SARS-CoV-2

COVID-19 is a disease caused by the SARS-CoV-2 virus and represents one of the greatest health problems that humanity faces at the moment. Therefore, efforts have been made with the objective of seeking therapies that could be effective in combating this problematic. In the search for ligands, computational chemistry plays an essential role, since it allows the screening of thousands of molecules on a given target, in order to save time and money for the in vitro or in vivo pharmacological stage. In this paper, we perform a virtual screening by docking looking for potential inhibitors of the NSP16-NSP10 protein dimer (methyltransferase) from SARS-CoV-2, by evaluating a homemade databank of molecules found in plants of the Caatinga Brazilian biome, compounds from ZINC online molecular database, as well as structural analogues of the enzymatic cofactor s-adenosylmethionine (SAM) and a known inhibitor in the literature, sinefungin (SFG), provided at PubChem database. All the evaluated sets presented molecules that deserve attention, highlighting four compounds from ZINC as the most promising ligands. These results contribute to the discovery of new molecular hits, in the search of potential agents against SARS-CoV-2 virus, still unveiling a pathway that can be used in combined therapies.

Interfering with mRNA Methylation by the 2′O-Methyltransferase (NSP16) from SARS-CoV-2 to Tackle the COVID-19 Disease

The pandemic associated to Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has resulted in a huge number of deaths and infected people. Although several vaccine programmes are currently underway and have reached phase 3, and a few small size drugs repurposed to aid treatment of severe cases of COVID-19 infections, effective therapeutic options for this disease do not currently exist. NSP16 is a S-adenosyl-L-Methionine (SAM) dependent 2′O-Methyltransferase that converts mRNA cap-0 into cap-1 structure to prevent virus detection by cell innate immunity mechanisms. NSP16 methylates the ribose 2′O-position of the first nucleotide of the mRNA only in the presence of an interacting partner, the protein NSP10. This feature suggests that inhibition of the NSP16 may represent a therapeutic window to treat COVID-19. To test this idea, we performed comparative structural analyses of the NSP16 present in human coronaviruses and developed a sinefungin (SFG) similarity-based virtual screening campaign to assess the druggability of the SARS-CoV-2 NSP16 enzyme. Through these studies, we identified the SFG analogue 44601604 as a promising more potent inhibitor of NSP16 to limit viral replication in infected cells, favouring viral clearance.

Structural insight into the recognition of S-adenosyl-L-homocysteine and sinefungin in SARS-CoV-2 Nsp16/Nsp10 RNA cap 2′-O-Methyltransferase

The recent ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to rapidly spread across the world. To date, neither a specific antiviral drug nor a clinically effective vaccine is available. Among the 15 viral non-structural proteins (nsps), nsp16 methyltransferase has been considered as a potential target due to its crucial role in RNA cap 2′-O-methylation process, preventing the virus detection by cell innate immunity mechanisms. In the present study, molecular recognition between the two natural nucleoside analogs (S-adenosyl-l-homocysteine (SAH) and sinefungin (SFG)) and the SARS-CoV-2 nsp16/nsp10/m7GpppAC5 was studied using all-atom molecular dynamics simulations and free energy calculations based on MM/GBSA and WaterSwap approaches. The binding affinity and the number of hot-spot residues, atomic contacts, and H-bond formations of SFG/nsp16 complex were distinctly higher than those of SAH/nsp16 system, consistent with the lower water accessibility at the enzyme active site. Notably, only SFG could electrostatically interact with the 2′-OH and N3 of RNA’s adenosine moiety, mimicking the methyl transfer reaction of S-adenosyl-l-methionine substrate. The atomistic binding mechanism obtained from this work paves the way for further optimizations and designs of more specific SARS-CoV-2 nsp16 inhibitors in the fight against COVID-19.

Genomewide Analysis of Mode of Action of the S-Adenosylmethionine Analogue Sinefungin in Leishmania infantum.

To further our understanding of one-carbon metabolism in the protozoan parasite Leishmania, we conducted genomic screens to study how the parasite responded to sinefungin (SNF) selection. SNF is a structural analogue of S-adenosylmethionine (AdoMet), a key methyl group donor to a number of biomolecules. One screen consisted of sequencing SNF-resistant mutants generated by stepwise selection with gradually increasing drug concentrations. These studies demonstrated deletion of the AdoMet transporter (AdoMetT1) by intergenic recombination as a crucial loss-of-function marker for SNF resistance. The second screen consisted of Cos-seq, a gain-of-function cosmid-based genomewide functional screen with increasing SNF concentration coupled to next-generation sequencing. Cosmids enriched in that screen and sequenced led to the identification of (i) the AdoMet synthetase (METK) as the major SNF target, (ii) an mRNA [(guanine-N7)-methyltransferase (CMT1)], (iii) a leucine carboxyl methyltransferase (LCMT), (iv) two tryparedoxin genes, and (v) two protein phosphatase regulatory genes. Further functional exploration indicated that LCMT interacts with one phosphatase catalytic subunit (PP2AC) and that mutation of the C-terminal leucine residue of PP2AC affects sinefungin susceptibility. These holistic screens led to the identification of transporters, biosynthetic genes, RNA and protein methyltransferases, as well as phosphatases linked to AdoMet-mediated functions in Leishmania IMPORTANCE The two main cellular metabolic one-carbon donors are reduced folates and S-adenosylmethionine, whose biosynthetic pathways have proven highly effective in chemotherapeutic interventions in various cell types. Sinefungin, a nucleoside analogue of S-adenosylmethionine, was shown to have potent activity against the protozoan parasite Leishmania Here, we studied resistance to sinefungin using whole-genome approaches as a way to further our understanding of the role of S-adenosylmethionine in this parasite and to reveal novel potential drug targets. These approaches allowed the characterization of novel features related to S-adenosylmethionine function in Leishmania which could further help in the development of sinefungin-like compounds against this pathogenic parasite.

A Mass Spectrometric Assay of METTL3/METTL14 Methyltransferase Activity.

A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA processing, stability, and translation. Methylation of adenosine at the N6 position (m6A) in messenger RNA (mRNA) is currently the most well-studied RNA modification and is catalyzed by the RNA methyltransferase complex METTL3/METTL14. Once generated, m6A can modulate mRNA splicing, export, localization, degradation, and translation. Although potent and selective inhibitors exist for several members of the Type I S-adenosylmethionine (SAM)-dependent methyltransferase family, no inhibitors have been reported for METTL3/METTL14 to date. To facilitate drug discovery efforts, a sensitive and robust mass spectrometry-based assay for METTL3/METTL14 using self-assembled monolayer desorption/ionization (SAMDI) technology has been developed. The assay uses an 11-nucleotide single-stranded RNA compared to a previously reported 27-nucleotide substrate. IC50 values of mechanism-based inhibitors S-adenosylhomocysteine (SAH) and sinefungin (SFG) are comparable between the SAMDI and radiometric assays that use the same substrate. This work demonstrates that SAMDI technology is amenable to RNA substrates and can be used for high-throughput screening and compound characterization for RNA-modifying enzymes.

Design, synthesis and in vitro anti-Zika virus evaluation of novel Sinefungin derivatives

We report herein the design and synthesis of a series of novel Sinefungin (SIN) derivatives, based on the structures of SIN and its analogue EPZ004777. Our results reveal that target compounds 1ad-af, 1ba-bb and 1bf-bh show better activity (IC50 = 4.56–20.16 μM) than EPZ004777 (IC50 = 35.19 μM). Surprisingly, SIN was founded to be not as active (IC50 > 50 μM) as we and other research groups predicted. Interestingly, the intermediates 9a-b and 11b display potent anti-ZIKV potency (IC50 = 6.33–29.98 μM), and compound 9a also exhibits acceptable cytotoxicity (CC50 > 200 μM), suggesting their promising potential to be leads for further development.

Structural basis of arginine asymmetrical dimethylation by PRMT6.

PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine mark on proteins such as histone H3R2. Asymmetric dimethylation of histone H3R2 by PRMT6 acts as a repressive mark that antagonizes trimethylation of H3 lysine 4 by the MLL histone H3K4 methyltransferase. PRMT6 is overexpressed in several cancer types, including prostate, bladder and lung cancers; therefore, it is of great interest to develop potent and selective inhibitors for PRMT6. Here, we report the synthesis of a potent bisubstrate inhibitor GMS [6'-methyleneamine sinefungin, an analog of sinefungin (SNF)], and the crystal structures of human PRMT6 in complex, respectively, with S-adenosyl-L-homocysteine (SAH) and the bisubstrate inhibitor GMS that shed light on the significantly improved inhibition effect of GMS on methylation activity of PRMT6 compared with SAH and an S-adenosyl-L-methionine competitive methyltransferase inhibitor SNF. In addition, we also crystallized PRMT6 in complex with SAH and a short arginine-containing peptide. Based on the structural information here and available in the PDB database, we proposed a mechanism that can rationalize the distinctive arginine methylation product specificity of different types of arginine methyltransferases and pinpoint the structural determinant of such a specificity.

Crystal structure of Rv2258c from Mycobacterium tuberculosis H37Rv, an S -adenosyl- l -methionine-dependent methyltransferase

The Mycobacterium tuberculosis Rv2258c protein is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase). Here, we have determined its crystal structure in three forms: a ligand-unbound form, a binary complex with sinefungin (SFG), and a binary complex with S-adenosyl-L-homocysteine (SAH). The monomer structure of Rv2258c consists of two domains which are linked by a long α-helix. The N-terminal domain is essential for dimerization and the C-terminal domain has the Class I MTase fold. Rv2258c forms a homodimer in the crystal, with the N-terminal domains facing each other. It also exists as a homodimer in solution. A DALI structural similarity search with Rv2258c reveals that the overall structure of Rv2258c is very similar to small-molecule SAM-dependent MTases. Rv2258c interacts with the bound SFG (or SAH) in an extended conformation maintained by a network of hydrogen bonds and stacking interactions. Rv2258c has a relatively large hydrophobic cavity for binding of the methyl-accepting substrate, suggesting that bulky nonpolar molecules with aromatic rings might be targeted for methylation by Rv2258c in M. tuberculosis. However, the ligand-binding specificity and the biological role of Rv2258c remain to be elucidated due to high variability of the amino acid residues defining the substrate-binding site.

Abstract 2437: Crystal structures of CARM1 bound to sinefungin and diverse peptide substrates

Abstract Co-activator-associated arginine methyltransferase 1 (CARM1) is a protein arginine N-methyltransferase (PRMT) enzyme that has been implicated in a variety of cancers including AML (1) and breast (2), prostate (3), lung (4) and colorectal (5) carcinomas. CARM1 is known to methylate H3 histones and non-histone substrates including p300/CBP, AIB1/SRC-3 and PABP1 (6). To date, several crystal structures of CARM1 have been solved, including structures with small molecule inhibitors (7), but no ternary structures with nucleotide and peptide substrate have been reported. Here, the crystal structures of human CARM1 with the SAM mimic sinefungin (SFG) and three different peptide sequences from histone H3 and PAPB1 are presented, and both non-methylated and singly-methylated arginine residues are exemplified. Extensive interactions are seen with residues Glu266, Glu257, and His414 and the substrate arginine side chain. Two key hydrogen bonds are made by the side chain of Asn161 with the backbone carbonyl of the P’1 peptide residue and the backbone NH of the P’3 peptide residue. The carbonyl of the P1 peptide residue engages the protein through water mediated hydrogen bonds. These structures show how the CARM1 binding site is capable of accommodating a variety of peptide sequences. Comparisons to known CARM1 complexes with small molecules provide additional insights into inhibitor design. 1. Vu, L. P. et al, PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex. Cell Rep 2013, 5 (6), 1625-38. 2. Al-Dhaheri, M. et al, CARM1 is an important determinant of ERalpha-dependent breast cancer cell differentiation and proliferation in breast cancer cells. Cancer Res 2011, 71 (6), 2118-28. 3. Kim, Y. R. et al, Differential CARM1 expression in prostate and colorectal cancers. BMC Cancer 2010, 10, 197. 4. Elakoum, R. et al, CARM1 and PRMT1 are dysregulated in lung cancer without hierarchical features. Biochimie 2014, 97, 210-8. 5. Ou, C. Y. et al, A coactivator role of CARM1 in the dysregulation of beta-catenin activity in colorectal cancer cell growth and gene expression. Mol Cancer Res 2011, 9 (5), 660-70. 6. Bedford M.T.; Clarke S.G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 2009, 33, 1-13. 7. Sack, J.S et al, Structural basis for CARM1 inhibition by indole and pyrazole inhibitors. Biochem J 2011, 436, 331-9. Citation Format: Ann Boriack-Sjodin, Lei Jin, Suzanne L. Jacques, Allison Drew, Margaret Porter Scott, Scott Ribich, Oscar Moradei. Crystal structures of CARM1 bound to sinefungin and diverse peptide substrates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2437. doi:10.1158/1538-7445.AM2015-2437

Refolding of a fully functional flavivirus methyltransferase revealed that S-adenosyl methionine but not S-adenosyl homocysteine is copurified with flavivirus methyltransferase.

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2'-O positions of the viral RNA cap by using S-adenosyl-l-methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by-product S-adenosyl-l-homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM-analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.

Two-stage gene assembly/cloning of a member of the TspDTI subfamily of bifunctional restriction endonucleases, TthHB27I

The Thermus sp. family of bifunctional type IIS/IIG/IIC restriction endonucleases (REase)–methyltransferases (MTase) comprises thermo-stable TaqII, TspGWI, TspDTI, TsoI, Tth111II/TthHB27I enzymes as well as a number of putative enzymes/open reading frames (ORFs). All of the family members share properties including a large protein size (ca. 120kDa), amino acid (aa) sequence homologies, enzymatic activity modulation by S-adenosylmethionine (SAM), recognition of similar asymmetric cognate DNA sites and cleavage at a distance of 11/9 nt. Analysis of the enzyme aa sequences and domain/motif organisation led to further Thermus sp. family division into the TspDTI and TspGWI subfamilies. The latter exhibits an unprecedented phenomenon of DNA recognition change upon substitution of SAM by its analogue, sinefungin (SIN), towards a very frequent DNA cleavage. We report cloning in Escherichia coli (E. coli), using a two-stage procedure and a putative tthHB27IRM gene, detected by bioinformatics analysis of the Thermus thermophilus HB27 (T. thermophilus) genome. The functionality of a 3366 base pair (bp)-/1121 aa-long, high GC content ORF was validated experimentally through the expression in E. coli. Protein features corroborated with the reclassification of TthHB27I into the TspDTI subfamily, which manifested in terms of aa-sequence/motif homologies and insensitivity to SIN-induced specificity shift. However, both SAM and SIN stimulated the REase DNA cleavage activity by at least 16–32 times; the highest was observed for the Thermus sp. family. The availability of TthHB27I and the need to include SAM or SIN in the reaction in order to convert the enzyme from “hibernation” status to efficient DNA cleavage is of practical significance in molecular biotechnology, extending the palette of available REase specificities.

Genetic mapping reveals that sinefungin resistance in Toxoplasma gondii is controlled by a putative amino acid transporter locus that can be used as a negative selectable marker.

Quantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasite Toxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDR(r) and type 10 VAND-SNF(r). The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of the SNR1 gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies in T. gondii.

Cofactor analogue-induced chemical reactivation of endonuclease activity in a DNA cleavage/methylation deficient TspGWI N473A variant in the NPPY motif

We reported previously that TspGWI, a prototype enzyme of a new Thermus sp. family of restriction endonucleases-methyltransferases (REases-MTases), undergoes the novel phenomenon of sinefungin (SIN)-caused specificity transition. Here we investigated mutant TspGWI N473A, containing a single amino acid (aa) substitution in the NPPY motif of the MTase. Even though the aa substitution is located within the MTase polypeptide segment, DNA cleavage and modification are almost completely abolished, indicating that the REase and MTase are intertwined. Remarkably, the TspGWI N473A REase functionality can be completely reconstituted by the addition of SIN. We hypothesize that SIN binds specifically to the enzyme and restores the DNA cleavage-competent protein tertiary structure. This indicates the significant role of allosteric effectors in DNA cleavage in Thermus sp. enzymes. This is the first case of REase mutation suppression by an S-adenosylmethionine (SAM) cofactor analogue. Moreover, the TspGWI N473A clone strongly affects E. coli division control, acting as a ‘selfish gene’. The mutant lacks the competing MTase activity and therefore might be useful for applications in DNA manipulation. Here we present a case study of a novel strategy for REase activity/specificity alteration by a single aa substitution, based on the bioinformatic analysis of active motif locations, combining (a) aa sequence engineering (b) the alteration of protein enzymatic properties, and (c) the use of cofactor–analogue cleavage reconstitution and stimulation.

The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity

Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN) increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca2+, SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca2+, substrate or both) and MTase state (in the presence of SIN and substrate, SIN and product or product alone). Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca2+ and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.

S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.

A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries

BackgroundGenomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scission methods are consequently needed. An alternative method for obtaining higher quality DNA fragments involves partial digestion with restriction endonucleases (REases).We have shown previously that class-IIS/IIC/IIG TspGWI REase, the prototype member of the Thermus sp. enzyme family, can be chemically relaxed by a cofactor analogue, allowing it to recognize very short DNA sequences of 3-bp combined frequency. Such frequently cleaving REases are extremely rare, with CviJI/CviJI*, SetI and FaiI the only other ones found in nature. Their unusual features make them very useful molecular tools for the development of representative DNA libraries.ResultsWe constructed a horse genomic library and a deletion derivative library of the butyrylcholinesterase cDNA coding region using a novel method, based on TaqII, Thermus sp. family bifunctional enzyme exhibiting cofactor analogue specificity relaxation. We used sinefungin (SIN) – an S-adenosylmethionine (SAM) analogue with reversed charge pattern, and dimethylsulfoxide (DMSO), to convert the 6-bp recognition site TaqII (5′-GACCGA-3′ [11/9]) into a theoretical 2.9-bp REase, with 70 shortened variants of the canonical recognition sequence detected. Because partial DNA cleavage is an inherent feature of the Thermus sp. enzyme family, this modified TaqII is uniquely suited to quasi-random library generation.ConclusionsIn the presence of SIN/DMSO, TaqII REase is transformed from cleaving every 4096 bp on average to cleaving every 58 bp. TaqII SIN/DMSO thus extends the palette of available REase prototype specificities. This phenomenon, employed under partial digestion conditions, was applied to quasi-random DNA fragmentation. Further applications include high sensitivity probe generation and metagenomic DNA amplification.