SAM-dependent Methyltransferase Research Articles

Giardia fibrillarin: a bioinformatics exploration of sequence and structure.

Fibrillarin is a key nucleolar S-adenosyl-L-methionine (SAM)-dependent methyltransferase, highly conserved among species in both sequence and function. It is actively involved in numerous cellular processes, particularly in the early stages of pre-ribosomal RNA processing. Although a fibrillarin orthologue has been identified in Giardia, an intestinal protozoan parasite responsible for numerous infections worldwide, its functional and structural features in this parasite remain largely unexplored. In this study, bioinformatics tools were used to analyze the sequence and structure of Giardia fibrillarin to provide insights that could aid future experimental and therapeutic investigations. Taking advantage of the protein sequence of Giardia fibrillarin, multiple bioinformatics tools were employed to estimate its domains, nuclear and nucleolar localization signals (NLS and NoLS), post-translational modifications, phylogeny, and three-dimensional structure. The analysis revealed significant conservation of Giardia fibrillarin, showing close relationships with archaeal and parasitic amoeba orthologs. It retains essential features such as the SAM-dependent methyltransferase domain, a glycine- and arginine-rich domain, and both NLS and NoLS. While the core region of the protein is structurally conserved, the N-terminal region exhibits notable divergence. The potential for inhibition of Giardia fibrillarin by SAM competitors suggests that it could be a promising target for drug development, particularly for strains resistant to current treatments.

The Second Methylation in Psilocybin Biosynthesis Is Enabled by a Hydrogen Bonding Network Extending into the Secondary Sphere Surrounding the Methyltransferase Active Site.

The Psilocybe cubensis SAM-dependent methyltransferase, PsiM, catalyzes the last step in the biosynthesis of psilocybin. Likely evolved from monomethylating RNA methyltransferases, PsiM acquired a key amino acid exchange in the secondary sphere of the active site, M247 N, which is responsible for its capacity to dimethylate. Two variants, PsiMN247M and PsiMN247A, were generated to further examine the role of Asn247 for mono- and dimethylation in PsiM. Herein, we present the kinetic profiles of both variants and crystal structures at resolutions between 0.9 and 1.0 Å. Each variant was crystallized as a ternary complex with the non-methylated acceptor substrate, norbaeocystin and S-adenosyl-l-homocysteine, and in a second complex with the cofactor analog, sinefungin, and the monomethylated substrate, baeocystin. Consistent with the inability of the variants to catalyze a second methyl transfer, these structures reveal catalytically non-productive conformations and a high level of disorder of the methylamine group of baeocystin. Additionally, both variants exhibit destabilization in the β5-β7 sheets and a conserved β-turn of the core Rossmann fold, causing 20-fold reduced substrate binding and 2-fold lower catalytic efficiency even with norbaeocystin. Our structural and kinetic analyses of the variants suggest that Asn247 is essential to allow enough space in the active site for multiple methylations while also participating in a network of hydrogen bonds that stabilizes secondary structure elements in the immediate vicinity of the active site for optimal methylation of norbaeocystin.

New Insights Into TRMT10A Syndrome: Case Report and Literature Review.

TRMT10A is related to a syndrome characterized by early-onset diabetes mellitus, microcephaly, epilepsy, and intellectual disability. We report a case of a patient showing spastic-ataxic paraparesis and Dandy-Walker variant associated with a causative homozygous c.421-1G > A variant in the TRMT10A gene, affecting a canonical splicing site. This mutation disrupts the "SAM-dependent methyltransferase TRM10-type domain", which is implicated in methylation and S-adenosylmethionine metabolic biological processes, crucial for mitochondrial and glucose metabolism. The prominent neurological involvement of our patient enhances the implication of TRMT10A in the brain development, suggesting a potential association between TRMT10A variants and dominant neurological phenotypes. This case expands the clinical spectrum of TRMT10A syndrome highlighting the importance of considering this gene in the evaluation of patients with brain/cerebellar malformations and spastic-ataxic paraparesis. Further research is warranted to elucidate the underlying pathogenic mechanisms and potential therapeutic implications.

Targeting Leishmaniasis with Nitrovinyl Derivatives: Their Synthesis, In Vitro Assessment, and Computational Exploration.

Leishmaniasis is an affliction caused by the protozoan parasites of the Leishmania genus. This disease impacts a substantial global populace, exceeding one million individuals, leading to disability-adjusted life years and fatalities, particularly within tropical regions. At present, the existing drug therapies have not attained a degree of efficacy that can be unequivocally classified as genuinely triumphant. In this context, the conception of novel compounds possessing the capacity to impede the parasite's life cycle at various stages holds considerable significance. In this research endeavor, an exploration was undertaken involving the design and synthesis of nineteen derivatives incorporating the nitrovinyl pharmacophore. The subsequent evaluation of their impacts on L. major was conducted through a combination of in vitro (amastigote and promastigote inhibition) and in silico (molecular docking) investigations. All of the compounds were synthesized and purified with good yields. In the amastigote inhibition assay, compounds 10, 15, and 18 showed better inhibitory effects than the standard drug meglumine antimonate (MA). Regarding the synergistic impact of synthesized compounds and MA together, all outcomes were significantly better than those of monotherapy of each in amastigote and macrophage forms. In the promastigote assay, compounds 2, 8, 12, 15, 16, 17, and 19 demonstrated superior inhibitory effects compared to MA. Moreover, compounds 4, 12, and 15 showed the best synergies with MA in inhibiting amastigotes. According to docking scores, 1XTP (a SAM-dependent methyltransferase) and 4G5D (Prostaglandin F synthase) receptors were found to be the most probable targets in their mechanism of action. In vitro evaluations and computational analyses strongly suggest that these compounds could be effective against both L. major amastigotes and promastigotes. Additionally, they exhibited notable synergistic interactions with MA against both living forms of the parasite.

Mangrove fungi in action: Novel bioremediation strategy for high-chloride wastewater

Bioremediation of extremely high-chloride wastewater poses significant challenges due to the adverse effects of elevated salt concentrations on most microorganisms, where chloride levels can be as high as 7% (w/v). Mangrove wetlands derived fungus, Aspergillus aculeatus, emerged as a promising candidate, capable of removing approximately 40% of chloride ions in environments with concentration of 15% (w/v), representative of industrial wastewater conditions. Transcriptomics and biochemical assays conducted under increasing salt conditions revealed that elevated chloride concentrations induce the expression and activity of S-adenosyl methionine-dependent methyltransferase, which facilitates the conversion of chloride into chloromethane. This is the first report characterizing the biological mechanism behind high salt tolerance and chloride removal capacity of Aspergillus aculeatus. This salt remediation mechanism may work as a starter for developing future bioremediation strategies to treat high-chloride wastewater using fungi, offering an eco-friendly alternative to traditional physical or chemical methods.

Functional investigation of the SAM-dependent methyltransferase RdmB in anthracycline biosynthesis

A novel sub-class of S-adenosyl-l-methionine (SAM)-dependent methyltransferases catalyze atypical chemical transformations in the biosynthesis of anthracyclines. Exemplified by RdmB from Streptomyces purpurascens, it was found with 10-decarboxylative hydroxylation activity on anthracyclines. We herein investigated the catalytic activities of RdmB and discovered a previously unknown 4-O-methylation activity. The site-directed mutagenesis studies proved that the residue at position R307 and N260 are vital for the decarboxylative hydroxylation and 4-O-methylation, respectively, which define two distinct catalytic centers in RdmB. Furthermore, the multifunctionality of RdmB activity was found as cofactor-dependent and stepwise. Our findings expand the versatility and importance of methyltransferases and should aid studies to enrich the structural diversity and bioactivities of anthracyclines.

An epigenetically mediated double negative cascade from EFD to HB21 regulates anther development

Epigenetic modifications are crucial for plant development. EFD (ExineFormationDefect) encodes a SAM-dependent methyltransferase that is essential for the pollen wall pattern formation and male fertility in Arabidopsis. In this study, we find that the expression of DRM2, a de novo DNA methyltransferase in plants, complements for the defects in efd, suggesting its potential de novo DNA methyltransferase activity. Genetic analysis indicates that EFD functions through HB21, as the knockout of HB21 fully restores fertility in efd mutants. DNA methylation and histone modification analyses reveal that EFD represses the transcription of HB21 through epigenetic mechanisms. Additionally, we demonstrate that HB21 directly represses the expression of genes crucial for pollen formation and anther dehiscence, including CalS5, RPG1/SWEET8, CYP703A2 and NST2. Collectively, our findings unveil a double negative regulatory cascade mediated by epigenetic modifications that coordinates anther development, offering insights into the epigenetic regulation of this process.

Structural Basis of Substrate Recognition by the Postmitosane Modification Enzyme MitM in Mitomycin Biosynthesis.

Mitomycins make up a class of natural molecules produced by Streptomyces with strong antibacterial and antitumor activities. MitM is a key postmitosane modification enzyme involved in mitomycin biosynthesis in Streptomyces caespitosus. This protein was previously suggested to catalyze the aziridinium methylation of mitomycin A and the mitomycin intermediate 9a-demethyl-mitomycin A as an N-methyltransferase. The structural basis for MitM to recognize cofactor S-adenosyl-l-methionine (SAM) and substrate mitomycin A is unknown. Here, we determined the crystal structures of apo-MitM and MitM-mitomycin A-S-adenosylhomocysteine (SAH) ternary complexes with resolutions of 2.23 and 2.80 Å, respectively. We found that MitM adopts a class I SAM-dependent methyltransferase fold and forms a homodimer in solution. Conformational changes in a series of residues involved in the formation of active pockets assist MitM in binding SAH and mitomycin A. In particular, the 28ALGAASLGE36 loop changes most significantly. When mitomycin A binds, the bending direction of this loop is reversed, changing the entrance of the active site from open to closed. This study provides structural insights into MitM's involvement in the postmitosane stage of mitomycin biosynthesis and provides a template for the engineering of methyltransferases.

Characterization of a unique catechol-O-methyltransferase as a molecular drug target in parasitic filarial nematodes.

Filarial nematodes cause severe illnesses in humans and canines including limb deformities and disfigurement, heart failure, blindness, and death, among others. There are no vaccines, and current drugs against filarial nematodes infections have only modest effects and are prone to complications. We identified a gene (herein called DiMT) encoding an S-adenosyl-L-methionine (SAM)-dependent methyltransferase with orthologs in parasite filarial worms but not in mammals. By in silico analysis, DiMT possesses catalytic sites for binding SAM and catecholamines with high affinity. We expressed and purified recombinant DiMT protein and used it as an enzyme in a series of SAM-dependent methylation assays. DiMT acted specifically as a catechol-O-methyltransferase (COMT), catalyzing catabolic methylation of dopamine, and depicted Michaelis Menten kinetics on substrate and co-substrate. Among a set of SAM-dependent methyltransferase inhibitors, we identified compounds that bound with high affinity to DiMT's catalytic sites and inhibited its enzymatic activity. By testing the efficacy of DiMT inhibitors against microfilariae of Dirofilaria immitis in culture, we identified three inhibitors with concentration- and time-dependent effect of killing D. immitis microfilariae. Importantly, RNAi silencing of a DiMT ortholog in Caenorhabditis elegans has been shown to be lethal, likely as a result of excessive accumulation of active catecholamines that inhibit worm locomotion, pharyngeal pumping and fecundity. Together, we have unveiled DiMT as an essential COMT that is conserved in parasitic filarial nematodes, but is significantly different from mammalian COMTs and, therefore, is a viable target for development of novel drugs against filarial nematode infections.

Biosynthesis of the Non-canonical C17 Sesquiterpenoids Chlororaphen A and B from Pseudomonas chlororaphis.

Chlororaphens A and B are structurally unique non-canonical C17 sesquiterpenoids from Pseudomonas chlororaphis that are made by two SAM-dependent methyltransferases and a type I terpene synthase. This study addresses the mechanism of their formation in isotopic labelling experiments and DFT calculations. The results demonstrate an astonishing complexity with distribution of labellings within a cyclopentane core that is reversely connected to two acyclic fragments in chlororaphen A and B. In addition, the uptake of up to 14 deuterium atoms from D2O was observed. These findings are explainable by a repeated late stage multistep rearrangement sequence. The absolute configurations of the chlororaphens and their biosynthetic intermediates were elucidated in stereoselective labelling experiments.

Whole genome identification, molecular docking and expression analysis of enzymes involved in the selenomethionine cycle in Cardamine hupingshanensis

BackgroundThe selenomethionine cycle (SeMTC) is a crucial pathway for the metabolism of selenium. The basic bioinformatics and functions of four enzymes involved in the cycle including S-adenosyl-methionine synthase (MAT), SAM-dependent methyltransferase (MTase), S-adenosyl-homocysteine hydrolase (SAHH) and methionine synthase (MTR), have been extensively reported in many eukaryotes. The identification and functional analyses of SeMTC genes/proteins in Cardamine hupingshanensis and their response to selenium stress have not yet been reported.ResultsIn this study, 45 genes involved in SeMTC were identified in the C. hupingshanensis genome. Phylogenetic analysis showed that seven genes from ChMAT were clustered into four branches, twenty-seven genes from ChCOMT were clustered into two branches, four genes from ChSAHH were clustered into two branches, and seven genes from ChMTR were clustered into three branches. These genes were resided on 16 chromosomes. Gene structure and homologous protein modeling analysis illustrated that proteins in the same family are relatively conserved and have similar functions. Molecular docking showed that the affinity of SeMTC enzymes for selenium metabolites was higher than that for sulfur metabolites. The key active site residues identified for ChMAT were Ala269 and Lys273, while Leu221/231 and Gly207/249 were determined as the crucial residues for ChCOMT. For ChSAHH, the essential active site residues were found to be Asn87, Asp139 and Thr206/207/208/325. Ile204, Ser111/329/377, Asp70/206/254, and His329/332/380 were identified as the critical active site residues for ChMTR. In addition, the results of the expression levels of four enzymes under selenium stress revealed that ChMAT3-1 genes were upregulated approximately 18-fold, ChCOMT9-1 was upregulated approximately 38.7-fold, ChSAHH1-2 was upregulated approximately 11.6-fold, and ChMTR3-2 genes were upregulated approximately 28-fold. These verified that SeMTC enzymes were involved in response to selenium stress to varying degrees.ConclusionsThe results of this research are instrumental for further functional investigation of SeMTC in C. hupingshanensis. This also lays a solid foundation for deeper investigations into the physiological and biochemical mechanisms underlying selenium metabolism in plants.

Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis.

Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a bfaA gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. Mycobacterium avium subspecies paratuberculosis (Map) growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including Mycobacterium smegmatis. While Map C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type Map strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by bfaB and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the bfaA gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type Map bfaA knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of Map and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for Map.IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the Mycobacterium genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for Mycobacterium tuberculosis. This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, Map strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of Mycobacterium avium subspecies paratuberculosis produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of Map. This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid.

Biotransformation of Bioactive Metabolites in Cassiae Semen by Endogenous Enzymes and Probiotics.

The present study evaluated the potential of endogenous enzymes and probiotics in transforming bioactive metabolites to reduce the purgative effect and improve the functional activity of Cassiae Semen and verified and revealed the biotransformation effect of endogenous enzymes. Although probiotics, especially Lactobacillus rhamnosus, exerted the transformation effect, the endogenous enzymes proved to be more effective in transforming the components of Cassiae Semen. After biotransformation by endogenous enzymes for 12 h, the levels of six anthraquinones in Cassiae Semen increased by at least 2.98-fold, and free anthraquinones, total phenolics, and antioxidant activity also showed significant improvement, accompanied by an 82.2% reduction in combined anthraquinones responsible for the purgative effect of Cassiae Semen. Further metabolomic analysis revealed that the biotransformation effect of endogenous enzymes on the bioactive metabolites of Cassiae Semen was complex and diverse, and the biotransformation of quinones and flavonoids was particularly prominent and occurred by three primary mechanisms, hydrolyzation, methylation, and dimerization, might under the action of glycosyl hydrolases, SAM-dependent methyltransferases, and CYP450s. Accordingly, biotransformation by endogenous enzymes emerges as a mild, economical, food safety risk-free, and effective strategy to modify Cassiae Semen into an excellent functional food.

Comparative Analysis of Two NNMT Bisubstrate Inhibitors through Chemoproteomic Studies: Uncovering the Role of Unconventional SAM Analogue Moiety for Improved Selectivity.

Unconventional S-adenosyl-L-methionine (SAM) mimics with enhanced hydrophobicity are an adaptable building block to develop cell-potent inhibitors for SAM-dependent methyltransferases as targeted therapeutics. We recently discovered cell-potent bisubstrate inhibitors for nicotinamide N-methyltransferase (NNMT) by using an unconventional SAM mimic. To delve into the selectivity implications of the unconventional SAM mimic, we employed a chemoproteomic approach to assess two potent NNMT inhibitors LL320 (Ki,app = 6.8 nM) and II399 (containing an unconventional SAM mimic, Ki,app = 5.9 nM) within endogenous proteomes. Our work began with the rational design and synthesis of immobilized probes 1 and 2, utilizing LL320 and II399 as parent compounds. Systematic analysis of protein networks associated with these probes revealed a comprehensive landscape. Notably, NNMT emerged as the top-ranking hit, substantiating the high selectivity of both inhibitors. Meanwhile, we identified additional interacting proteins for LL320 (38) and II399 (17), showcasing the intricate selectivity profiles associated with these compounds. Subsequent experiments confirmed LL320's interactions with RNMT, DPH5, and SAHH, while II399 exhibited interactions with SHMT2 and MEPCE. Importantly, incorporating the unconventional SAM mimic in II399 led to improved selectivity compared to LL320. Our findings underscore the importance of selectivity profiling and validate the utilization of the unconventional SAM mimic as a viable strategy to create highly selective and cell-permeable inhibitors for SAM-dependent methyltransferases.

Betaine attenuates age-related suppression in autophagy via Mettl21c/p97/VCP axis to delay muscle loss

Age-related impairment of autophagy accelerates muscle loss and lead to sarcopenia. Betaine can delay muscle loss as a dietary methyl donor via increasing S-adenosyl-L-methionine (SAM, a crucial metabolite for autophagy regulation) in methionion cycle. However, whether betaine can regulate autophagy level to attenuate degeneration in aging muscle remains unclear. Herein, male C57BL/6J young mice (YOU, 2-month-old), old mice (OLD, 15-month-old), and 2%-betaine-treated old mice (BET, 15-month-old) were employed and raised for 12 weeks. All mice underwent body composition examination and grip strength test before being sacrificed. Betaine alleviated age-related decline in muscle mass and strength. Meanwhile, betaine preserved the expression autophagy markers (Atg5, Atg7, LC3-II, and Beclin1) both at transcriptional and translational level during the aging process. RNA-sequencing results generated from mice gastrocnemius muscle found Mettl21c, a SAM-dependent autophagy-regulating methyltransferase, was significantly higher expressed in BET and YOU group. Results were further validated by qPCR and western bloting. In vitro, C2C12 cells with or without Mettl21c RNA interference were treated different concentration of betaine (0 mM, 10 mM) under methionine-starved condition. Compared with control group, betaine upregulated autophagy markers expression and autophagy flux. By increasing the SAM level, betaine facilitated trimethylation of p97 (Mettl21c downstream effector) into valosin-containing protein (VCP). Increased VCP promoted autophagic turnover of cellular components, ATP production, and cell differentiation. Knock-down of Metthl21c dismissed improvements mentioned above. Collectively, betaine could enhance aged skeletal muscle autophagy level via Mettl21c/p97/VCP axis to delay muscle loss.

Application of the C3-Methyltransferase StspM1 for the Synthesis of the Natural Pyrroloindole Motif.

Even though pyrroloindoles are widely present in natural products with different kinds of biological activities, their selective synthesis remains challenging with existing tools in organic chemistry, and there is furthermore a demand for stereoselective and mild methods to access this structural motif. Nature uses C3-methyltransferases to form the pyrroloindole framework, starting from the amino acid tryptophan. In the present study, the SAM-dependent methyltransferase StspM1 from Streptomyces sp. HPH0547 is used to build the pyrroloindole structural motif in tryptophan-based diketopiperazines (DKP). The substrate scope of the enzyme regarding different Trp-Trp-DKP isomers was investigated on an experimental and computational level. After further characterization and optimization of the methylation reaction with a design of experiment approach, a preparative scale reaction with the immobilized enzyme including a SAM regeneration system was performed to show the synthetic use of this biocatalytic tool to access the pyrroloindole structural motif.

De novo drug designing coupled with brute force screening and structure guided lead optimization gives highly specific inhibitor of METTL3: a potential cure for Acute Myeloid Leukaemia

Expression of METTL3, a SAM dependent methyltransferase, which deposits m6A on mRNA is linked to poor prognosis in Acute Myeloid Leukaemia and other type of cancers. Down regulation of this epitranscriptomic regulator has been found to inhibit cancer progression. Silencing the methyltransferase activity of METTL3 is a lucrative strategy to design anticancer drugs. In this study 3600 commercially available molecules were screened against METTL3 using brute force screening approach. However, none of these compounds take advantage of the unique Y-shaped binding cavity of the protein, raising the need for de novo drug designing strategies. As such, 125 branched, Y-shaped molecules were designed by “stitching” together the chemical fragments of the best inhibitors that interact strongly with the METTL3 binding pocket. This results in molecules that have the three-dimensional structure and functional groups which enable it to fit in the METTL3 cavity like fingers in a glove, having unprecedented selectivity and binding affinities. The designed compounds were further refined based on Lipinski’s rule, docking score and synthetic accessibility. The molecules faring well in these criteria were simulated for 100 ns to check the stability of the protein inhibitor complex followed by binding free energy calculation.Communicated by Ramaswamy H. Sarma

In silico Design of Novel SAM Analogs as Potential Inhibitors Against N2G966 16s rRNA Methyltransferase (RsmD)

Introduction: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), has its natural history tracing back to 70,000 years. Latent M.tb infection is the reservoir of the TB epidemic. M.tb is becoming more prevalent and acquiring multidrug resistance among the first-line antibiotics. Methods: Methylation is one of the main mechanisms through which bacteria develop resistance, hence targeting methyltransferases provides the opportunity to achieve two-birds-with-one-stone: a) antibiotic: inhibiting the translation activity and b) anti-resistance: eliminating methylation as a mode of resistance. Currently, no known drugs or lead molecules are targeting the methyltransferases, in general, and rRNA Small Subunit Methyltransferase D (RsmD) family, in particular, in M.tb species. Results and Discussion: S-Adenosyl-L-methionine(SAM) is known as the universal donor of a methyl group which is an indispensable cofactor for the proper functioning of SAM-dependent methyltransferases. This in silico study attempts to design and develop novel SAM-analog inhibitors against RsmD, which in turn affects the growth and survival of M.tb in TB patients. The SAM-analogs were designed, after careful study and analysis of RsmD pharmacophore and SAM binding properties. The functional groups such as amide, amine, acetamide, formamide, hydroxyl, fluorine, iodine, and bromine were used to design novel analogs with the aim to improve the binding of analog with RsmD. The analogs that gave better docking scores followed by favourable binding affinities and ADMET properties than native SAM were ranked. Conclusion: Among the library of SAM analogs, the top two analogs with IDs: SAM_172 and SAM_153 need testing and validation for their efficacy through in vitro and in vivo studies.

A Universal, Continuous Assay for SAM-dependent Methyltransferases.

Enzyme-catalyzed late-stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S-adenosyl-l-methionine-dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo- and regioselective methylation of O-, N-, S- and C-atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence-based, high-throughput assay for SAM-dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S-adenosyl-l-homocysteine into H2 S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O-MTs and an N-MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates.

Methylation in hangtaimycin biosynthesis and its antibacterial activities

About two-thirds of small molecule drugs contain methyl group and it plays a very important role in the drug development. So, methyltransferases catalyzing the methylation have always attracted great attention. Hangtaimycin (HTM) is a potent hepatoprotective agent. Previous study showed that its biosynthetic gene cluster contained three methyltransferase domains, but their characteristics in HTM biosynthetic pathway has not been revealed. In this study, we clarified multi-methylations in HTM biosynthesis in vivo. It showed that the two S-adenosylmethionine-dependent methyltransferases (SAM-MTs) of HtmA2(-module 6)-MT domain and HtmB2(-module 18)-MT domain are responsible for the installation of methyl group at C-45 and N-12, respectively, whereas the FK506 methyltransferase (FKMT) type O-methyltransferase of HtmB1(-module 16)-MT domain take care of the methylation at O-21 of HTM. We also reported the antibacterial activities of HTM in this study, and found that it showed activities against M. luteus, B. thuringiensis and A. baumannii with MIC of 4 μg/mL, 4 μg/mL, and 64 μg/mL, respectively.