The sisomicin-gentamicin resistance methylase gene (sgm) from Micromonospora zionensis (the producer of antibiotic G-52 [6-N-methyl-sisomicin]) encodes an enzyme that modifies 16S rRNA and thereby confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides. Here, we report that this gene is regulated on the translational level. The Escherichia coli lacZ gene and operon fusion system was used, and it was shown that an extra copy of the sgm gene decreases the activity of the fusion protein. These results suggested that expression of the sgm gene is regulated by the translational autorepression because of binding of the methylase to its own mRNA. It was shown by computer analysis that the same hexanucleotide (CCGCCC) is present 14 bp before the ribosome-binding site and in the C-1400 region of 16S rRNA, i.e., the region in which most of the aminoglycosides act. A deletion that removes the hexanucleotide before the gene fusion is not prone to negative autoregulation. This mode of regulation of the sgm gene ensures that enough methylase molecules protect the cell from the action of its own antibiotic. On the other hand, if all of the ribosomes are modified, Sgm methylase binds to its own mRNA in an autorepressive manner.
The 16S rRNA methyltransferase Sgm from “Micromonospora zionensis” confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-l-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a KD (binding constant) of 17.6 μM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.
The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-l-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles.
The sisomicin-gentamicin resistance methylase (sgm) gene was isolated from Micromonospora zionensis and cloned in Streptomyces lividans. The sgm gene was expressed in Micromonospora melanosporea, where its own promoter was active, and also in Escherichia coli under the control of the lacZ promoter. The complete nucleotide sequence of 1,122 bp and a transcription start point were determined. The sequence contains an open reading frame that encodes a polypeptide of 274 amino acids. The methylation of 30S ribosomal subunits by Sgm methylase accounts adequately for all known resistance characteristics of M. zionensis, but expression of high-level resistance to hygromycin B is background dependent. A comparison of the amino acid sequence of the predicted Sgm protein with the deduced amino acid sequences for the 16S rRNA methylases showed extensive similarity of Grm and significant similarity to KgmB but not to KamB methylase.
The aminoglycoside resistance genes sgm from Micromonospora zionensis and kgmB from Streptomyces tenebrarius were cloned into a yeast expression vector to test whether the encoded prokaryotic methylases can modify the 18S rRNA A-site and thus confer resistance to G-418. Despite the detectable presence of mRNAs in yeast cells, neither G-418-resistant yeast transformants nor positive western blot signals were obtained. Neither methylase was capable of methylating 40S subunits despite very high conservation of the antibiotic rRNA binding sites. However, the results provide novel insight into the action of Sgm by showing that it methylates the same site as KgmB, i.e. G1405 in 16S rRNA.
Aminoglycoside resistance; 16S rRNA methylases; Site of action; G1405; Expression in yeast
NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin–apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics.
Arm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants of Escherichia coli, corresponding to the genotypes rsmF+, ΔrsmF, rsmF+
rmtC+, and ΔrsmF rmtC+. When analyzed for the antimicrobial resistance pattern, the ΔrsmF bacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility in E. coli. Competition experiments between the isogenic E. coli strains showed that, contrary to expectation, acquisition of rmtC does not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.
Methyltransferases use S-adenosylmethionine (AdoMet) as methyl group donor, forming S-adenosylhomocysteine (AdoHcy) and methylated substrates, including DNA and proteins. AdoHcy inhibits most methyltransferases. Accumulation of intracellular AdoHcy secondary to Hcy elevation elicits global DNA hypomethylation. We aimed at determining the extent at which protein arginine methylation status is affected by accumulation of intracellular AdoHcy. AdoHcy accumulation in human umbilical vein endothelial cells was induced by inhibition of AdoHcy hydrolase by adenosine-2,3-dialdehyde (AdOx). As a measure of protein arginine methylation status, the levels of monomethylarginine (MMA) and asymmetric and symmetric dimethylated arginine residues (ADMA and SDMA, respectively) in cell protein hydrolysates were measured by HPLC. A 10% decrease was observed at a 2.5-fold increase of intracellular AdoHcy. Western blotting revealed that the translational levels of the main enzymes catalyzing protein arginine methylation, protein arginine methyl transferases (PRMTs) 1 and 5, were not affected by AdoHcy accumulation. Global DNA methylation status was evaluated by measuring 5-methylcytosine and total cytosine concentrations in DNA hydrolysates by LC-MS/MS. DNA methylation decreased by 10% only when intracellular AdoHcy concentration accumulated to 6-fold of its basal value. In conclusion, our results indicate that protein arginine methylation is more sensitive to AdoHcy accumulation than DNA methylation, pinpointing a possible new player in methylation-related pathology.
High-level resistance to a broad spectrum of aminoglycoside antibiotics can arise through either N7-methyl guanosine 1405 (m7G1405) or N1-methyl adenosine 1408 (m1A1408) modifications at the drug binding site in the bacterial 30S ribosomal subunit decoding center. Two distinct families of 16S ribosomal RNA (rRNA) methyltransferases that incorporate these modifications were first identified in aminoglycoside-producing bacteria but were more recently identified in both human and animal pathogens. These resistance determinants thus pose a new threat to the usefulness of aminoglycosides as antibiotics, demanding urgent characterization of their structures and activities. Here, we describe approaches to cloning, heterologous expression in E. coli, and purification of two A1408 rRNA methyltransferases: KamB from the aminoglycoside-producer Streptoalloteichus tenebrarius and NpmA identified in a clinical isolate of pathogenic E. coli ARS3. Antibiotic minimum inhibitory concentration (MIC) assays and in vitro analysis of KamB and NpmA using circular dichroism (CD) spectroscopy, S-adenosyl-L-methionine (SAM) binding by isothermal titration calorimetry and 30S subunit methylation assays showed both enzymes were soluble, folded and active. Finally, crystals of each enzyme complexed with SAM were obtained, including selenomethionine-derived KamB, that will facilitate high-resolution X-ray crystallographic analyses of these important bacterial antibiotic-resistance determinants.
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.
The enzyme protein carboxyl methyltransferase type II has been recently shown to play a crucial role in the repair of damaged proteins. S-adenosylmethionine (AdoMet) is the methyl donor of the reaction, and its demethylated product, S-adenosylhomocysteine (AdoHcy), is the natural inhibitor of this reaction, as well as of most AdoMet-dependent methylations. We examined erythrocyte membrane protein methyl esterification in chronic renal failure (CRF) patients on conservative treatment or hemodialyzed to detect possible alterations of the methylation pattern, in a condition where a state of disrupted red blood cell function is present. We observed a significant reduction in membrane protein methyl esterification in both groups, compared to control. The decrease was particularly evident for cytoskeletal component ankyrin, which is known to be involved in membrane stability and integrity. Moreover, we observed a severalfold rise in AdoHcy levels, while AdoMet concentration was comparable to that detected in the control, resulting in a lower [AdoMet]/[AdoHcy] ratio (P < 0.001). Our findings show an impairment of this posttranslational modification of proteins, associated with high AdoHcy intracellular concentration in CRF. The data are consistent with the notion that, in CRF, structural damages accumulate in erythrocyte membrane proteins, and are not adequately repaired.
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.
O-linked methylation of sugar substituents is a common modification in the biosynthesis of many natural products, and is catalyzed by multiple families of S-adenosyl-L-methioine (SAM or AdoMet) dependent methyltransferases. Mycinamicins, potent antibiotics from Micromonospora griseorubida, can be methylated at two positions on a 6-deoxyallose substituent. The first methylation is catalyzed by MycE, a SAM- and metal-dependent methyltransferase. Crystal structures were determined for MycE bound to the product S-adenosyl-L-homocysteine (SAH or AdoHcy) and magnesium, both with and without the natural substrate, mycinamicin VI. This represents the first structure of a natural product sugar methyltransferase in complex with its natural substrate. MycE is a tetramer of a two-domain polypeptide, comprising a C-terminal catalytic methyltransferase domain and an N-terminal auxiliary domain, which is important for quaternary assembly and for substrate binding. The symmetric MycE tetramer has a novel methyltransferase organization in which each of the four active sites is formed at the junction of three monomers within the tetramer. The active site structure supports a mechanism in which a conserved histidine acts as a general base, and the metal ion helps to position the methyl acceptor, and to stabilize a hydroxylate intermediate. A conserved tyrosine is suggested to support activity through interactions with the transferred methyl group from the SAM methyl donor. The structure of the free enzyme reveals a dramatic order-disorder transition in the active site relative to the SAH complexes, suggesting a mechanism for product/substrate exchange through concerted movement of five loops and the polypeptide C-terminus.
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.
► AdoHcy is a potent product inhibitor of AdoMet-dependent methyltransferases. ► AdoHcy accumulates in hyperhomocysteinemia. ► Yeast is an advantageous system to understand AdoHcy toxicity. ► Lipid metabolism is deregulated in response to AdoHcy accumulation.
AdoMet; AdoHcy; Homocysteine; S-adenosyl-L-homocysteine hydrolase
The target cytosines of (cytosine-5)-DNA methyltransferases in prokaryotic and eukaryotic DNA show increased rates of C-->T transition mutations compared to non-target cytosines. These mutations are induced either by the spontaneous deamination of 5-mC-->T generating inefficiently repaired G:T rather than G:U mismatches, or by the enzyme-induced C-->U deamination which occurs under conditions of reduced levels of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy). We tested whether various inhibitors of (cytosine-5)-DNA methyltransferases analogous to AdoMet and AdoHcy would affect the rate of enzyme-induced deamination of the target cytosine by M.HpaII and M.SssI. Interestingly, we found two compounds, sinefungin and 5'-amino-5'-deoxyadenosine, that increased the rate of deamination 10(3)-fold in the presence and 10(4)-fold in the absence of AdoMet and AdoHcy. We have therefore identified the first mutagenic compounds specific for the target sites of (cytosine-5)-DNA methyltransferases. A number of analogs of AdoMet and AdoHcy have been considered as possible antiviral, anticancer, antifungal and antiparasitic agents. Our findings show that chemotherapeutic agents with affinities to the cofactor binding pocket of (cytosine-5)-DNA methyltransferase should be tested for their potential mutagenic effects.
Homocysteine (Hcy) has been recognized for the past five decades as a risk factor for atherosclerosis. However, the role of Hcy in the pathological changes associated with atherosclerosis as well as the pathological mechanisms triggered by Hcy accumulation is
poorly understood. Due to the reversal of the physiological direction of the reaction catalyzed by S-adenosyl-L-homocysteine
hydrolase Hcy accumulation leads to the synthesis of S-adenosyl-L-homocysteine (AdoHcy). AdoHcy is a strong product
inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases, and to date more than 50 AdoMet-dependent
methyltransferases that methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids have been
identified. Phospholipid methylation is the major consumer of AdoMet, both in mammals and in yeast. AdoHcy accumulation induced
either by Hcy supplementation or due to S-adenosyl-L-homocysteine hydrolase deficiency results in inhibition of phospholipid
methylation in yeast. Moreover, yeast cells accumulating AdoHcy also massively accumulate triacylglycerols (TAG). Similarly, Hcy
supplementation was shown to lead to increased TAG and sterol synthesis as well as to the induction of the unfolded protein
response (UPR) in mammalian cells. In this review a model of deregulation of lipid metabolism in response to accumulation of
AdoHcy in Hcy-associated pathology is proposed.
S-Adenosylmethionine (AdoMet) is a ubiquitous cofactor in biomethylations and, in that role, becomes S-adenosylhomocysteine (AdoHcy), which serves as a biofeedback inhibitor of the methylation process. In seeking to avail unexplored structural variations of AdoHcy for biological studies, its 6′-oxa analog and two corresponding carbocyclic nucleosides (based on aristeromycin and neplanocin) have been prepared via common convergent syntheses.
S-adenosylhomocysteine; aristeromycin; neplanocin; Schöllkopf auxiliary
Sisomicin is a new antibiotic produced by Micromonospora inyoensis. The in vitro activities of sisomicin, gentamicin, and tobramycin, three similar aminoglycosides, were determined against 228 clinical isolates representing 10 genera of common pathogens. No difference was noted in the activities of these antimicrobial agents when assayed by a standard broth dilution technique against Klebsiella, Enterobacter, Escherichia, Salmonella, Citrobacter, enterococci, or Staphylococcus aureus. Sisomicin was significantly more active than tobramycin against Serratia and indole-positive Proteus strains. Sisomicin was significantly more active than gentamicin against indole-negative Proteus strains and slightly more active against indole-positive Proteus strains. Tobramycin was more active than sisomicin or gentamicin against Pseudomonas and indole-negative Proteus strains. Gram-negative bacilli resistant to one of the three antimicrobial agents were not necessarily resistant to either of the other two. Activity of sisomicin was independent of the susceptibility or resistance of these isolates to nine other antimicrobial agents as assayed by the Bauer-Kirby technique. The presence of 50% human serum did not antagonize the in vitro activity of sisomicin against gram-negative isolates. Because sisomicin showed certain advantages over gentamicin or tobramycin in vitro, further investigation of this new antimicrobial agent is warranted.
Hyperhomocysteinemia and factors of homocysteine metabolism, S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet), may play a role in Alzheimer’s disease (AD). With liquid-chromatography-tandem-mass-spectrometry AdoMet and AdoHcy were determined in brains of 8- and 15-month-old APP/PS1 Alzheimer mice, and their possible roles in AD brains investigated. The finding that AdoMet levels do not differ between the genotypes in (young) 8-month-old mice, but are different in (older) 15-month-old APP/PS1 mice compared to their wild-type littermates, suggests that alterations in AdoMet are a consequence of AD pathology rather than a cause. During aging, AdoMet levels decreased in the brains of wild-type mice, whereas AdoHcy levels diminished in both wild type and APP/PS1 mice. The finding that AdoMet levels in APP/PS1 mice are not decreased during aging (in contrast to wild-type mice), is probably related to less demand due to neurodegeneration. No effect of the omega-3 fatty acid docosahexaenoic acid (DHA) or cholesterol-enriched diets on AdoMet or AdoHcy levels were found.
S-adenosylmethionine; S-adenosylhomocysteine; Alzheimer’s disease; APP/PS1 mice; Cholesterol; DHA
DNA (cytosine-5)-methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 5-position of cytosine residues and thereby silence transcription of regulated genes. DNMTs are important epigenetic targets. However, isolated DNMTs are weak catalysts and are difficult to assay. We report an ultrasensitive luciferase-linked continuous assay that converts the S-adenosyl-L-homocysteine product of DNA methylation to a quantifiable luminescent signal. Results with this assay are compared with the commonly used DNA labeling from [methyl-3H]AdoMet. A 5′-methylthioadenosine-adenosylhomocysteine nucleosidase is used to hydrolyze AdoHcy to adenine. Adenine phosphoribosyl transferase converts adenine to AMP and pyruvate orthophosphate dikinase converts AMP to ATP. Firefly luciferase gives a stable luminescent signal that results from continuous AMP recycling to ATP. This assay exhibits a broad dynamic range (0.1–1000 pmol of AdoHcy). The rapid response time permits continuous assays of DNA methylation detected by light output. The assay is suitable for high-throughput screening of chemical libraries with DNMT activity. The kinetic properties of human and bacterial CpG methyltransferases are characterized using this assay. Human catalytic domain DNMT3b activation by DNMT3L is shown to involve two distinct kinetic states that alter kcat but not Km for AdoMet. The assay is shown to be robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine.
DNA methyltransferase; DNMT; CpG islands; luciferase; epigenetics; S-adenosyl-L-homocysteine
S-Adenosylmethionine (AdoMet or SAM)-dependent methyltransferases belong to a large and diverse family of group-transfer enzymes that perform vital biological functions on a host of substrates. Despite the progress in genomics, structural proteomics and computational biology, functional annotation of methyltransferases remains a challenge. Herein, we report the synthesis and activity of a new AdoMet analog functionalized with a ketone group. Using catechol O-methyltransferase (COMT, EC 184.108.40.206) and thiopurine S-methyltransferase (TPMT, EC 220.127.116.11) as model enzymes, this robust and readily accessible analog displays kinetic parameters that are comparable to AdoMet and exhibits multiple turnovers with enzyme. More importantly, this AdoMet surrogate displays the same substrate specificity as the natural methyl donor. Incorporation of the ketone group allows for subsequent modification via bio-orthogonal labeling strategies and sensitive detection of the tagged ketone products. Hence, this AdoMet analog expands the toolbox available to interrogate the biochemical functions of methyltransferases.
The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)·S-adenosyl-l-methionine (AdoMet) complex was determined at 2.45-Å resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal “L30-like” putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr·rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.
Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m5U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m5U modifications. However, as previously shown, the m5U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m5U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m5U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.
A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.
This paper reports the third proven human case of deficient S-adenosylhomocysteine (AdoHcy) hydrolase activity. The patient is similar to the only two previously reported cases with this disorder in having severe myopathy, developmental delay, elevated serum creatine kinase (CK) concentrations, and hypermethioninaemia. Although he has been followed from infancy, the basic enzyme deficiency was established only at age 26 years. The diagnosis was based on markedly elevated plasma concentrations of both AdoHcy and S-adenosylmethionine, some 20% of the mean control activity of AdoHcy hydrolase activity in haemolysates of his red-blood cells, and two missense mutations in his gene encoding AdoHcy hydrolase. He had low values of erythrocyte phosphatidylcholine and plasma free choline and marginally elevated excretion of guanidinoacetate, suggesting that the elevated AdoHcy may have been inhibiting methylation of phosphatidylethanolamine and guanidinoacetate. His leukocyte DNA was globally more methylated than the DNA's of his parents or the mean extent of methylation measured in age-matched control subjects.