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1.  Novel Plasmid-Mediated 16S rRNA m1A1408 Methyltransferase, NpmA, Found in a Clinically Isolated Escherichia coli Strain Resistant to Structurally Diverse Aminoglycosides▿  
Antimicrobial Agents and Chemotherapy  2007;51(12):4401-4409.
We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.
doi:10.1128/AAC.00926-07
PMCID: PMC2168023  PMID: 17875999
2.  Structural insights into the function of aminoglycoside-resistance A1408 16S rRNA methyltransferases from antibiotic-producing and human pathogenic bacteria 
Nucleic Acids Research  2010;38(21):7791-7799.
X-ray crystal structures were determined of the broad-spectrum aminoglycoside-resistance A1408 16S rRNA methyltransferases KamB and NpmA, from the aminoglycoside-producer Streptoalloteichus tenebrarius and human pathogenic Escherichia coli, respectively. Consistent with their common function, both are Class I methyltransferases with additional highly conserved structural motifs that embellish the core SAM-binding fold. In overall structure, the A1408 rRNA methyltransferase were found to be most similar to a second family of Class I methyltransferases of distinct substrate specificity (m7G46 tRNA). Critical residues for A1408 rRNA methyltransferase activity were experimentally defined using protein mutagenesis and bacterial growth assays with kanamycin. Essential residues for SAM coenzyme binding and an extended protein surface that likely interacts with the 30S ribosomal subunit were thus revealed. The structures also suggest potential mechanisms of A1408 target nucleotide selection and positioning. We propose that a dynamic extended loop structure that is positioned adjacent to both the bound SAM and a functionally critical structural motif may mediate concerted conformational changes in rRNA and protein that underpin the specificity of target selection and activation of methyltransferase activity. These new structures provide important new insights that may provide a starting point for strategies to inhibit these emerging causes of pathogenic bacterial resistance to aminoglycosides.
doi:10.1093/nar/gkq627
PMCID: PMC2995053  PMID: 20639535
3.  Detection of Methyltransferases Conferring High-Level Resistance to Aminoglycosides in Enterobacteriaceae from Europe, North America, and Latin America▿  
The alteration of ribosomal targets by recently described 16S rRNA methyltransferases confers resistance to most aminoglycosides, including arbekacin. Enterobacteriaceae and nonfermentative bacilli acquired through global surveillance programs were screened for the presence of these enzymes on the basis of phenotypes that were resistant to nine tested aminoglycosides. Subsequent molecular studies determined that 20 of 21 (95.2%) methyltransferase-positive isolates consisted of novel species records or geographic occurrences (North America [armA and rmtB], Latin America [rmtD], and Europe [armA]; rmtA, rmtC, and npmA were not detected). The global emergence of high-level aminoglycoside resistance has become a rapidly changing event requiring careful monitoring.
doi:10.1128/AAC.01477-07
PMCID: PMC2346617  PMID: 18347105
4.  Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m7G methyltransferases 
Nucleic Acids Research  2010;38(12):4120-4132.
Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 Å resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m7G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.
doi:10.1093/nar/gkq122
PMCID: PMC2896518  PMID: 20194115
5.  Protein Arginine Methylation Is More Prone to Inhibition by S-Adenosylhomocysteine than DNA Methylation in Vascular Endothelial Cells 
PLoS ONE  2013;8(2):e55483.
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.
doi:10.1371/journal.pone.0055483
PMCID: PMC3568140  PMID: 23408989
6.  Expression, purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies 
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.
doi:10.1016/j.pep.2010.07.005
PMCID: PMC2966526  PMID: 20667473
7.  S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase 
PLoS ONE  2013;8(10):e76900.
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.
doi:10.1371/journal.pone.0076900
PMCID: PMC3793912  PMID: 24130807
8.  A Conformational Switch in the Active Site of BT_2972, a Methyltransferase from an Antibiotic Resistant Pathogen B. thetaiotaomicron 
PLoS ONE  2011;6(11):e27543.
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.
doi:10.1371/journal.pone.0027543
PMCID: PMC3225368  PMID: 22140448
9.  Enzymatic methyl esterification of erythrocyte membrane proteins is impaired in chronic renal failure. Evidence for high levels of the natural inhibitor S-adenosylhomocysteine. 
Journal of Clinical Investigation  1993;91(6):2497-2503.
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.
PMCID: PMC443310  PMID: 8514862
10.  Tissue-specific relationship of S-adenosylhomocysteine with allele-specific H19/Igf2 methylation and imprinting in mice with hyperhomocysteinemia 
Epigenetics  2013;8(1):44-53.
DNA methylation is linked to homocysteine metabolism through the generation of S-adenosylmethionine (AdoMet) and S-Adenosylhomocysteine (AdoHcy). The ratio of AdoMet/AdoHcy is often considered an indicator of tissue methylation capacity. The goal of this study is to determine the relationship of tissue AdoMet and AdoHcy concentrations to allele-specific methylation and expression of genomically imprinted H19/Igf2. Expression of H19/Igf2 is regulated by a differentially methylated domain (DMD), with H19 paternally imprinted and Igf2 maternally imprinted. F1 hybrid C57BL/6J x Castaneous/EiJ (Cast) mice with (+/−), and without (+/+), heterozygous disruption of cystathionine-β-synthase (Cbs) were fed a control diet or a diet (called HH) to induce hyperhomocysteinemia and changes in tissue AdoMet and AdoHcy. F1 Cast x Cbs+/− mice fed the HH diet had significantly higher plasma total homocysteine concentrations, higher liver AdoHcy, and lower AdoMet/AdoHcy ratios and this was accompanied by lower liver maternal H19 DMD allele methylation, lower liver Igf2 mRNA levels, and loss of Igf2 maternal imprinting. In contrast, we found no significant differences in AdoMet and AdoHcy in brain between the diet groups but F1 Cast x Cbs+/− mice fed the HH diet had higher maternal H19 DMD methylation and lower H19 mRNA levels in brain. A significant negative relationship between AdoHcy and maternal H19 DMD allele methylation was found in liver but not in brain. These findings suggest the relationship of AdoMet and AdoHcy to gene-specific DNA methylation is tissue-specific and that changes in DNA methylation can occur without changes in AdoMet and AdoHcy.
doi:10.4161/epi.23063
PMCID: PMC3549880  PMID: 23221482
homocysteine; genomic imprinting; DNA methylation; gene expression; H19; Igf2; tissue-specific; allele-specific
11.  Nucleophosmin Is Essential for Ribosomal Protein L5 Nuclear Export 
Molecular and Cellular Biology  2006;26(10):3798-3809.
Nucleophosmin (NPM/B23) is a key regulator in the regulation of a number of processes including centrosome duplication, maintenance of genomic integrity, and ribosome biogenesis. While the mechanisms underlying NPM function are largely uncharacterized, NPM loss results in severe dysregulation of developmental and growth-related events. We show that NPM utilizes a conserved CRM1-dependent nuclear export sequence in its amino terminus to enable its shuttling between the nucleolus/nucleus and cytoplasm. In search of NPM trafficking targets, we biochemically purified NPM-bound protein complexes from HeLa cell lysates. Consistent with NPM's proposed role in ribosome biogenesis, we isolated ribosomal protein L5 (rpL5), a known chaperone for the 5S rRNA. Direct interaction of NPM with rpL5 mediated the colocalization of NPM with maturing nuclear 60S ribosomal subunits, as well as newly exported and assembled 80S ribosomes and polysomes. Inhibition of NPM shuttling or loss of NPM blocked the nuclear export of rpL5 and 5S rRNA, resulting in cell cycle arrest and demonstrating that NPM and its nuclear export provide a unique and necessary chaperoning activity to rpL5/5S.
doi:10.1128/MCB.26.10.3798-3809.2006
PMCID: PMC1488996  PMID: 16648475
12.  Nucleophosmin Serves as a Rate-Limiting Nuclear Export Chaperone for the Mammalian Ribosome▿  
Molecular and Cellular Biology  2008;28(23):7050-7065.
Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.
doi:10.1128/MCB.01548-07
PMCID: PMC2593371  PMID: 18809582
13.  S-adenosyl-L-homocysteine hydrolase and methylation disorders: Yeast as a model system 
Biochimica et Biophysica Acta  2013;1832(1):204-215.
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.
Highlights
► 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.
doi:10.1016/j.bbadis.2012.09.007
PMCID: PMC3787734  PMID: 23017368
AdoMet; AdoHcy; Homocysteine; S-adenosyl-L-homocysteine hydrolase
14.  Methylation inhibitors can increase the rate of cytosine deamination by (cytosine-5)-DNA methyltransferase. 
Nucleic Acids Research  1996;24(16):3267-3275.
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.
PMCID: PMC146075  PMID: 8774911
15.  Characterizing DNA Methyltransferases With An Ultrasensitive Luciferase-Linked Continuous Assay 
Analytical chemistry  2011;83(12):4996-5004.
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.
doi:10.1021/ac200816m
PMCID: PMC3115440  PMID: 21545095
DNA methyltransferase; DNMT; CpG islands; luciferase; epigenetics; S-adenosyl-L-homocysteine
16.  Homocysteine as a Risk Factor for Atherosclerosis: Is Its Conversion to S-Adenosyl-L-Homocysteine the Key to Deregulated Lipid Metabolism? 
Journal of Lipids  2011;2011:702853.
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.
doi:10.1155/2011/702853
PMCID: PMC3151505  PMID: 21837278
17.  S-adenosylmethionine and S-adenosylhomocysteine levels in the aging brain of APP/PS1 Alzheimer mice 
Neurological Sciences  2009;30(5):439-445.
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.
doi:10.1007/s10072-009-0110-2
PMCID: PMC2746292  PMID: 19565184
S-adenosylmethionine; S-adenosylhomocysteine; Alzheimer’s disease; APP/PS1 mice; Cholesterol; DHA
18.  Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE* 
The Journal of biological chemistry  2006;282(8):5880-5887.
N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.
doi:10.1074/jbc.M608214200
PMCID: PMC2885967  PMID: 17189261
19.  6′-Oxa Analogs of S-Adenosylhomocysteine 
Tetrahedron  2012;68(1):65-71.
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.
doi:10.1016/j.tet.2011.10.063
PMCID: PMC3320718  PMID: 22489098
S-adenosylhomocysteine; aristeromycin; neplanocin; Schöllkopf auxiliary
20.  RecA-Mediated Gene Conversion and Aminoglycoside Resistance in Strains Heterozygous for rRNA 
Clinical resistance to aminoglycosides in general is due to enzymatic drug modification. Mutational alterations of the small ribosomal subunit rRNA have recently been found to mediate acquired resistance in bacterial pathogens in vivo. In this study we investigated the effect of 16S rRNA heterozygosity (wild-type [wt] and mutant [mut] operons at position 1408 [1408wt/1408mut]) on aminoglycoside resistance. Using an integrative vector, we introduced a single copy of a mutated rRNA operon (1408 A→G) into Mycobacterium smegmatis, which carries two chromosomal wild-type rRNA operons; the resultant transformants exhibited an aminoglycoside-sensitive phenotype. In contrast, introduction of the mutated rRNA operon into an M. smegmatis rrnB knockout strain carrying a single functional chromosomal wild-type rRNA operon resulted in aminoglycoside-resistant transformants. Subsequent analysis by DNA sequencing and RNase protection assays unexpectedly demonstrated a homozygous mutant genotype, rRNAmut/rRNAmut, in the resistant transformants. To investigate whether RecA-mediated gene conversion was responsible for the aminoglycoside-resistant phenotype in the rRNAwt/rRNAmut strains, recA mutant strains were generated by allelic exchange techniques. Transformation of the recA rrnB M. smegmatis mutant strains with an integrative vector expressing a mutated rRNA operon (Escherichia coli position 1408 A→G) resulted in transformants with an aminoglycoside-sensitive phenotype. Subsequent analysis showed stable heterozygosity at 16S rRNA position 1408 with a single wild-type allele and a single resistant allele. These results demonstrate that rRNA-mediated mutational resistance to aminoglycosides is recessive.
PMCID: PMC89142  PMID: 10049249
21.  Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi 
We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.
doi:10.1128/AAC.50.2.445-452.2006
PMCID: PMC1366916  PMID: 16436695
22.  Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase 
Nucleic Acids Research  2005;33(1):307-315.
DNA methylation plays important roles via regulation of numerous cellular mechanisms in diverse organisms, including humans. The paradigm bacterial methyltransferase (MTase) HhaI (M.HhaI) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-l-methionine (AdoMet) onto the target cytosine in DNA, yielding 5-methylcytosine and S-adenosyl-l-homocysteine (AdoHcy). The turnover rate (kcat) of M.HhaI, and the other two cytosine-5 MTases examined, is limited by a step subsequent to methyl transfer; however, no such step has so far been identified. To elucidate the role of cofactor interactions during catalysis, eight mutants of Trp41, which is located in the cofactor binding pocket, were constructed and characterized. The mutants show full proficiency in DNA binding and base-flipping, and little variation is observed in the apparent methyl transfer rate kchem as determined by rapid-quench experiments using immobilized fluorescent-labeled DNA. However, the Trp41 replacements with short side chains substantially perturb cofactor binding (100-fold higher KDAdoMet and KMAdoMet) leading to a faster turnover of the enzyme (10-fold higher kcat). Our analysis indicates that the rate-limiting breakdown of a long-lived ternary product complex is initiated by the dissociation of AdoHcy or the opening of the catalytic loop in the enzyme.
doi:10.1093/nar/gki175
PMCID: PMC546160  PMID: 15653631
23.  Homozygous Triplicate Mutations in Three 16S rRNA Genes Responsible for High-Level Aminoglycoside Resistance in Nocardia farcinica Clinical Isolates from a Canada-Wide Bovine Mastitis Epizootic▿  
Nocardia farcinica strains showing high-level resistance to amikacin were isolated from clinical cases in a Canada-wide bovine mastitis epizootic. Shotgun cloning of the resistance genes in the amikacin-resistant mastitis isolate N. farcinica IFM 10580 (W6220 [Centers for Disease Control and Prevention]) using a multicopy vector system revealed that the 16S rRNA gene with an A-to-G single-point mutation at position 1408 (in Escherichia coli numbering) conferred “moderate” cross-resistance to amikacin and other aminoglycosides to an originally susceptible N. farcinica strain IFM 10152. Subsequent DNA sequence analyses revealed that, in contrast to the susceptible strain, all three chromosomal 16S rRNA genes of IFM 10580, the epizootic clinical strain, contained the same A1408G point mutations. Mutant colonies showing high-level aminoglycoside resistance were obtained when the susceptible strain N. farcinica IFM 10152 was transformed with a multicopy plasmid carrying the A1408G mutant 16S rRNA gene and was cultured in the presence of aminoglycosides for 3 to 5 days. Of these transformants, at least two of the three chromosomal 16S rRNA genes contained A1408G mutations. A triple mutant was easily obtained from a strain carrying the two chromosomal A1408G mutant genes and one wild-type gene, even in the absence of the plasmid. The triple mutant showed the highest level of resistance to aminoglycosides, even in the absence of the plasmid carrying the mutant 16S rRNA gene. These results suggest that the homozygous mutations in the three 16S rRNA genes are responsible for the high-level aminoglycoside resistance found in N. farcinica isolates of the bovine mastitis epizootic.
doi:10.1128/AAC.00021-10
PMCID: PMC2876378  PMID: 20308368
24.  Approaches to measuring the activities of protein arginine N-methyltransferases 
Analytical biochemistry  2009;397(1):1-11.
Despite the emerging importance of protein arginine N-methyltransferase (PRMT) activity in regulating cellular processes, only a limited number of PRMT assays have been developed. Here, we compare several qualitative and quantitative methods that we use for measuring PRMT activity. Gel-based methods allow for the simultaneous detection of methyl transfer activity on multiple substrates, but require signals well above background in order to generate reliable data for quantitation, which can be challenging with low activity PRMTs or substrates that are poor methyl-acceptors. Techniques that measure S-adenosyl-L-homocysteine (AdoHcy) product formation suffer from a background caused by PRMT automethylation and the spontaneous formation of AdoHcy from S-adenosyl-L-methionine (AdoMet). However, when this background is controlled, this approach is useful for product inhibition studies. Methods that detect methylated arginines derived from acid hydrolysis of PRMT reaction samples can determine the absolute amounts of ω-NG-monomethylarginine (MMA), asymmetric ω-NG,NG-dimethylarginine (aDMA) or symmetric ω-NG,N′G-dimethylarginine (sDMA) to quantify PRMT activity. We describe separation methods of these methylated arginine derivatives by thin layer, reverse phase, or cation exchange chromatography, and quantification by radioactivity or mass spectrometry. The latter approach is advantageous because it does not require radiolabelled samples for detection, and activity is readily quantified with commercially available standards.
doi:10.1016/j.ab.2009.09.021
PMCID: PMC2808438  PMID: 19761747
PRMT; AdoMet; AdoHcy; methylarginine; enzyme kinetics
25.  A New Structural Form in the SAM/Metal-Dependent O-methyltransferase Family: MycE from the Mycinamycin Biosynthetic Pathway 
Journal of molecular biology  2011;413(2):438-450.
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.
doi:10.1016/j.jmb.2011.08.040
PMCID: PMC3193595  PMID: 21884704

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