- Biosynthesis of a thiamin antivitamin in clostridium botulinum
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Bacimethrin-derived 2′-methoxythiamin pyrophosphate inhibits microbial growth by disrupting metabolic pathways dependent on thiamin-utilizing enzymes. This study describes the discovery of the bacimethrin biosynthetic gene cluster of Clostridium botulinum A ATCC 19397 and in vitro reconstitution of bacimethrin biosynthesis from cytidine 5′-monophosphate.
- Cooper, Lisa E.,O'Leary, Seán E.,Begley, Tadhg P.
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- Photochemical deamination and demethylation of 5-methylcytosine
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Cytosine methylation is believed to play a pivotal role in eucaryotic cellular development as well as in viral latency. We have been investigating chemical mechanisms for the perturbation of methylation patterns, including the effects of ultraviolet radiation. We observed that, upon exposure to UV light, 5-methylcytosine (5mC) was converted to thymine, cytosine, and a series of 5-substituted cytosine derivatives as analyzed by gas chromatography/mass spectrometry. Deamination of 5mC to thymine proceeds via formation of the intermediate photohydrate. Formation of 5-substituted cytosine derivatives results from oxidation of the 5-methyl group with initial formation of 5-(hydroxymethyl)cytosine (hmC). Upon exposure to UV light, hmC is converted to cytosine. The conversion of hmC to cytosine likely results from photohydration and elimination of formaldehyde. It is proposed that endogenous oxidation and hydrolysis could result in demethylation of 5mC residues in DNA. Whereas hydrolytic deamination of 5mC to thymine has been widely discussed, demethylation of 5mC has not as yet been described.
- Privat, Eric,Sowers, Lawrence C.
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- Potent methyl oxidation of 5-methyl-2′-deoxycytidine by halogenated quinoid carcinogens and hydrogen peroxide via a metal-independent mechanism
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Halogenated quinones are a class of carcinogenic intermediates and are newly identified chlorination disinfection by-products in drinking water. We found recently that the highly reactive and biologically important hydroxyl radical (?OH) can be produced by halogenated quinones and H 2O2 independent of transition metal ions. However, it is not clear whether these quinoid carcinogens and H2O2 can oxidize the nucleoside 5-methyl-2′-deoxycytidine (5mdC) to its methyl oxidation products and, if so, what the underlying molecular mechanism is. Here we show that three methyl oxidation products, 5-(hydroperoxymethyl)-, 5-(hydroxymethyl)-, and 5-formyl-2′-deoxycytidine, could be produced when 5mdC was treated with tetrachloro-1,4-benzoquinone (TCBQ) and H 2O2. The formation of the oxidation products was markedly inhibited by typical ?OH scavengers and under anaerobic conditions. Analogous effects were observed with other halogenated quinones and the classic Fenton system. Based on these data, we propose that the oxidation of 5mdC by TCBQ/H2O2 might be through the following mechanism: ?OH produced by TCBQ/H2O2 may first abstract hydrogen from the methyl group of 5mdC, leading to the formation of 5-(2′-deoxycytidylyl)methyl radical, which may combine with O 2 to form the peroxyl radical. The unstable peroxyl radical transforms into the corresponding hydroperoxide 5-(hydroperoxymethyl)-2′- deoxycytidine, which reacts with TCBQ and results in the formation of 5-(hydroxymethyl)-2′-deoxycytidine and 5-formyl-2′-deoxycytidine. This is the first report that halogenated quinoid carcinogens and H 2O2 can induce potent methyl oxidation of 5mdC via a metal-independent mechanism, which may partly explain their potential carcinogenicity.
- Shao, Jie,Huang, Chun-Hua,Kalyanaraman, Balaraman,Zhu, Ben-Zhan
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- Efficient synthesis of 5-hydroxymethyl pyrimidines and their nucleosides using microwave irradiation
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Hydroxymethylation of uracil (1), cytosine (3), 5-hydroxymethyl-2′,3′-O-isopropylideneuridine (5), 5′-O-tert-butyldiphenylsilyl-2′,3′-O-isopropylideneuridine (7), 2′,3′-O-isopropylidenecytidine (9) and 2′,3′-O-isopropylidene-5′-O-tritylcytidine (11) was efficiently carried out with paraformaldehyde in alkaline medium under microwave irradiation in very high yield.
- Abdel-Rahman, Adel A.-H.,El Ashry, El Sayed H.
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- Excision of 5-Carboxylcytosine by Thymine DNA Glycosylase
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5-Methylcytosine (mC) is an epigenetic mark that is written by methyltransferases, erased through passive and active mechanisms, and impacts transcription, development, diseases including cancer, and aging. Active DNA demethylation involves TET-mediated stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), or 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and subsequent base excision repair. Many elements of this essential process are poorly defined, including TDG excision of caC. To address this problem, we solved high-resolution structures of human TDG bound to DNA with cadC (5-carboxyl-2′-deoxycytidine) flipped into its active site. The structures unveil detailed enzyme-substrate interactions that mediate recognition and removal of caC, many involving water molecules. Importantly, two water molecules contact a carboxylate oxygen of caC and are poised to facilitate acid-catalyzed caC excision. Moreover, a substrate-dependent conformational change in TDG modulates the hydrogen bond interactions for one of these waters, enabling productive interaction with caC. An Asn residue (N191) that is critical for caC excision is found to contact N3 and N4 of caC, suggesting a mechanism for acid-catalyzed base excision that features an N3-protonated form of caC but would be ineffective for C, mC, or hmC. We also investigated another Asn residue (N140) that is catalytically essential and strictly conserved in the TDG-MUG enzyme family. A structure of N140A-TDG bound to cadC DNA provides the first high-resolution insight into how enzyme-substrate interactions, including water molecules, are impacted by depleting the conserved Asn, informing its role in binding and addition of the nucleophilic water molecule.
- Pidugu, Lakshmi S.,Dai, Qing,Malik, Shuja S.,Pozharski, Edwin,Drohat, Alexander C.
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- Structure of the N-glycosidase MilB in complex with hydroxymethyl CMP reveals its Arg23 specifically recognizes the substrate and controls its entry
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5-Hydroxymethylcytosine (5hmC) is present in T-even phage and mammalian DNA as well as some nucleoside antibiotics, including mildiomycin and bacimethrin, during whose synthesis 5hmC is produced by the hydrolysis of 5-hydroxymethyl cytidine 5'-monophosphate (hmCMP) by an N-glycosidase MilB. Recently, the MilB-CMP complex structure revealed its substrate specificity for CMP over dCMP. However, hmCMP instead of CMP is the preferred substrate for MilB as supported by that its KM for CMP is ～27-fold higher than that for hmCMP. Here, we determined the crystal structures of MilB and its catalytically inactive E103A mutant in complex with hmCMP. In the structure of the complex, Phe22 and Arg23 are positioned in a cage-like active site resembling the binding pocket for the flipped 5-methylcytosine (5mC) in eukaryotic 5mC-binding proteins. Van der Waals interaction between the benzene ring of Phe22 and the pyrimidine ring of hmCMP stabilizes its binding. Remarkably, upon hmCMP binding, the guanidinium group of Arg23 was bent ～65° toward hmCMP to recognize its 5-hydroxymethyl group, inducing semi-closure of the cage-like pocket. Mutagenesis studies of Arg23 and bioinformatics analysis demonstrate that the positively charged Arg/Lys at this site is critical for the specific recognition of the 5-hydroxymethyl group of hmCMP. The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
- Zhao, Gong,Wu, Geng,Zhang, Yan,Liu, Guang,Han, Tiesheng,Deng, Zixin,He, Xinyi
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- Biomimetic Iron Complex Achieves TET Enzyme Reactivity**
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The epigenetic marker 5-methyl-2′-deoxycytidine (5mdC) is the most prevalent modification to DNA. It is removed inter alia via an active demethylation pathway: oxidation by Ten-Eleven Translocation 5-methyl cytosine dioxygenase (TET) and subsequent removal via base excision repair or direct demodification. Recently, we have shown that the synthetic iron(IV)-oxo complex [FeIV(O)(Py5Me2H)]2+ (1) can serve as a biomimetic model for TET by oxidizing the nucleobase 5-methyl cytosine (5mC) to its natural metabolites. In this work, we demonstrate that nucleosides and even short oligonucleotide strands can also serve as substrates, using a range of HPLC and MS techniques. We found that the 5-position of 5mC is oxidized preferably by 1, with side reactions occurring only at the strand ends of the used oligonucleotides. A detailed study of the reactivity of 1 towards nucleosides confirms our results; that oxidation of the anomeric center (1′) is the most common side reaction.
- Schmidl, David,Jonasson, Niko S. W.,Korytiaková, Eva,Carell, Thomas,Daumann, Lena J.
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- Functional succinate dehydrogenase deficiency is a common adverse feature of clear cell renal cancer
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Reduced succinate dehydrogenase (SDH) activity resulting in adverse succinate accumulation was previously considered relevant only in 0.05 to 0.5% of kidney cancers associated with germline SDH mutations. Here, we sought to examine a broader role for SDH loss in kidney cancer pathogenesis/progression. We report that underexpression of SDH subunits resulting in accumulation of oncogenic succinate is a common feature in clear cell renal cell carcinoma (ccRCC) (～80% of all kidney cancers), with a marked adverse impact on survival in ccRCC patients (n = 516). We show that SDH down-regulation is a critical brake in the TCA cycle during ccRCC pathogenesis and progression. In exploring mechanisms of SDH down-regulation in ccRCC, we report that Von Hippel-Lindau loss-induced hypoxia-inducible factor–dependent up-regulation of miR-210 causes direct inhibition of the SDHD transcript. Moreover, shallow deletion of SDHB occurs in ～20% of ccRCC. We then demonstrate that SDH loss-induced succinate accumulation contributes to adverse loss of 5-hydroxymethylcytosine, gain of 5-methylcytosine, and enhanced invasiveness in ccRCC via inhibition of ten-eleven translocation (TET)-2 activity. Intriguingly, binding affinity between the catalytic domain of recombinant TET-2 and succinate was found to be very low, suggesting that the mechanism of succinate-induced attenuation of TET-2 activity is likely via product inhibition rather than competitive inhibition. Finally, exogenous ascorbic acid, a TET-activating demethylating agent, led to reversal of the above oncogenic effects of succinate in ccRCC cells. Collectively, our study demonstrates that functional SDH deficiency is a common adverse feature of ccRCC and not just limited to the kidney cancers associated with germline SDH mutations.
- Aggarwal, Ritesh K.,Luchtel, Rebecca A.,Machha, Venkata,Tischer, Alexander,Zou, Yiyu,Pradhan, Kith,Ashai, Nadia,Ramachandra, Nandini,Albanese, Joseph M.,Yang, Jung-In,Wang, Xiaoyang,Aluri, Srinivas,Gordon, Shanisha,Aboumohamed, Ahmed,Gartrell, Benjamin A.,Hafizi, Sassan,Pullman, James,Shenoy, Niraj
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- TET-Like Oxidation in 5-Methylcytosine and Derivatives: A Computational and Experimental Study
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The epigenetic marker 5-methylcytosine (5mC) is an important factor in DNA modification and epigenetics. It can be modified through a three-step oxidation performed by ten-eleven-translocation (TET) enzymes and we have previously reported that the iron(IV)-oxo complex [Fe(O)(Py5Me2H)]2+ (1) can oxidize 5mC. Here, we report the reactivity of this iron(IV)-oxo complex towards a wider scope of methylated cytosine and uracil derivatives relevant for synthetic DNA applications, such as 1-methylcytosine (1mC), 5-methyl-iso-cytosine (5miC) and thymine (T/5mU). The observed kinetic parameters are corroborated by calculation of the C?H bond energies at the reactive sites which was found to be an efficient tool for reaction rate prediction of 1 towards methylated DNA bases. We identified oxidation products of methylated cytosine derivatives using HPLC-MS and GC-MS. Thereby, we shed light on the impact of the methyl group position and resulting C?H bond dissociation energies on reactivity towards TET-like oxidation.
- Jonasson, Niko S. W.,Jan?en, Rachel,Menke, Annika,Zott, Fabian L.,Zipse, Hendrik,Daumann, Lena J.
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p. 3333 - 3340
(2021/09/25)
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- 5-Methylcytosine is Oxidized to the Natural Metabolites of TET Enzymes by a Biomimetic Iron(IV)-Oxo Complex
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Ten-eleven-translocation (TET) methyl cytosine dioxygenases play a key role in epigenetics by oxidizing the epigenetic marker 5-methyl cytosine (5mC) to 5-hydroxymethyl cytosine (5hmC), 5-formyl cytosine (5fC), and 5-carboxy cytosine (5cC). Although much of the metabolism of 5mC has been studied closely, certain aspects—such as discrepancies among the observed catalytic activity of TET enzymes and calculated bond dissociation energies of the different cytosine substrates—remain elusive. Here, it is reported that the DNA base 5mC is oxidized to 5hmC, 5fC, and 5cC by a biomimetic iron(IV)-oxo complex, reminiscent of the activity of TET enzymes. Studies show that 5hmC is preferentially turned over compared with 5mC and 5fC and that this is in line with the calculated bond dissociation energies. The optimized syntheses of d3-5mC and d2-5hmC are also reported and in the reaction with the biomimetic iron(IV)-oxo complex these deuterated substrates showed large kinetic isotope effects, confirming the hydrogen abstraction as the rate-limiting step. Taken together, these results shed light on the intrinsic reactivity of the C?H bonds of epigenetic markers and the contribution of the second coordination sphere in TET enzymes.
- Jonasson, Niko S. W.,Daumann, Lena J.
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supporting information
p. 12091 - 12097
(2019/08/07)
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- Hydrophilic modifications in peptide nucleic acid - Synthesis and properties of PNA possessing 5-hydroxymethyluracil and 5-hydroxymethylcytosine
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We have investigated the chemistry for the incorporation of C5-hydroxymethyl-uracil and -cytosine in peptide nucleic acid (PNA) and the subsequent effect of this modification on PNA hybridization behavior. Largely based on literature precedent, we prepared a peptide nucleic acid monomer, possessing 5-hydroxymethyuracil, which was compatible with Fmoc-based oligopeptide synthesis. An improved, large-scale synthesis of 5-hydroxymethylcytosine was developed, as a starting point for the synthesis of a monomer containing this nucleobase. In each case, the hydroxyl group was blocked as a t-butyldiphenylsilyl ether, and the exocyclic amino group of cytosine was additionally blocked with the benzoyl-group. The modified monomers were incorporated into isolated positions in the oligomer sequence using standard protocols. The modified oligomers showed that the 5-hydroxymethyl group is compatible with triplex and duplex formation.
- Hudson, Robert H. E.,Liu, Yuhong,Wojciechowski, Filip
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p. 302 - 312
(2008/09/18)
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- Oxidation of nucleic acid related compounds by the peroxodisulfate ion
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The treatment of nucleic acid bases, nucleosides, and nucleotides with peroxodisulfate ion in a phosphate buffer solution at pH 7.0 or water at 70-75°C was investigated. The reaction of thymine and 5-methylcytosine nucleosides and nucleotides resulted in the oxidation of the 5-methyl groups. The oxidation products from 1,3-dimethyluracils and the time-course of the reaction of uracils led to two plausible reaction mechanisms for the oxidation of uracils.
- Itahara,Yoshitake,Koga,Nishino
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p. 2257 - 2264
(2007/10/02)
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- Oxidation of Cytosine and 5-Methylcytosine Nucleosides and 5-Methyl-2'-deoxycytidine 5'-Monophosphate with Peroxosulfate Ions
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Reaction of 5-methylcytosine nucleosides and nucleotide with Na2S2O8 resulted in an oxidation of the 5-methyl group, while treatment of them and cytosine nucleosides with KHSO5 gave the corresponding N3-oxides.
- Itahara, Toshio
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p. 1591 - 1594
(2007/10/02)
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- Nucleoside process
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Process for producing 1-halo-2-deoxy-2-fluoroarabinofuranoside derivatives bearing protective ester groups from 1,3,5-tri-O-acylribofuranose; the 1-halo compounds are intermediates in the synthesis of therapeutically active nucleosidic compounds.
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