- Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with Photocaging Groups and Reversible DNA Modification in Cascade Reactions
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Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site-specific inhibition and timed removal. S-Adenosyl-L-methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC-ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC-ChMAT at 1.87 ? revealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC-MATs were compatible with DNA- and RNA-MTases, enabling sequence-specific modification (“writing”) of plasmid DNA and light-triggered removal (“erasing”).
- Michailidou, Freideriki,Kl?cker, Nils,Cornelissen, Nicolas V.,Singh, Rohit K.,Peters, Aileen,Ovcharenko, Anna,Kümmel, Daniel,Rentmeister, Andrea
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- A sensitive mass spectrum assay to characterize engineered methionine adenosyltransferases with S-alkyl methionine analogues as substrates
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Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-l-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAMs) into the SAM analogues with engineered MATs. Here we describe a general sensitive activity assay for engineered MATs by converting the reaction products into S-alkylthioadenosines, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification. With this assay, 40 human MAT mutants were evaluated against 7 SAAMs as potential substrates. The structure-activity relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower Km value in contrast to native MATs), the gained activity toward the bulky SAAMs stems from their ability to maintain the desired linear SN2 transition state (reflected by higher kcat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAMs.
- Wang, Rui,Zheng, Weihong,Luo, Minkui
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- SELECTIVE INHIBITORS OF PROTEIN ARGININE METHYLTRANSFERASE 5 (PRMT5)
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The disclosure is directed to compounds of Formula (I) and Formula (II). Methods of their use and preparation is other described.
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Paragraph 00168
(2018/01/20)
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- The Catalytic Mechanism of the Class C Radical S-Adenosylmethionine Methyltransferase NosN
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S-Adenosylmethionine (SAM) is one of the most common co-substrates in enzyme-catalyzed methylation reactions. Most SAM-dependent reactions proceed through an SN2 mechanism, whereas a subset of them involves radical intermediates for methylating non-nucleophilic substrates. Herein, we report the characterization and mechanistic investigation of NosN, a class C radical SAM methyltransferase involved in the biosynthesis of the thiopeptide antibiotic nosiheptide. We show that, in contrast to all known SAM-dependent methyltransferases, NosN does not produce S-adenosylhomocysteine (SAH) as a co-product. Instead, NosN converts SAM into 5′-methylthioadenosine as a direct methyl donor, employing a radical-based mechanism for methylation and releasing 5′-thioadenosine as a co-product. A series of biochemical and computational studies allowed us to propose a comprehensive mechanism for NosN catalysis, which represents a new paradigm for enzyme-catalyzed methylation reactions.
- Ding, Wei,Li, Yongzhen,Zhao, Junfeng,Ji, Xinjian,Mo, Tianlu,Qianzhu, Haocheng,Tu, Tao,Deng, Zixin,Yu, Yi,Chen, Fener,Zhang, Qi
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supporting information
p. 3857 - 3861
(2017/03/27)
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- Organometallic Complex Formed by an Unconventional Radical S-Adenosylmethionine Enzyme
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Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAMCA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAMCA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron-nuclear double resonance (ENDOR) measurements with 13C and 2H isotopically labeled SAMCA support a π-complex between the C=C double bond of α-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM-related [4Fe-4S]+ cluster forming an organometallic complex with an alkene, shedding additional light on the mechanism of PhDph2 and expanding our current notions for the reactivity of [4Fe-4S] clusters in radical SAM enzymes.
- Dong, Min,Horitani, Masaki,Dzikovski, Boris,Pandelia, Maria-Eirini,Krebs, Carsten,Freed, Jack H.,Hoffman, Brian M.,Lin, Hening
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supporting information
p. 9755 - 9758
(2016/08/19)
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- Facile chemoenzymatic strategies for the synthesis and utilization of S-adenosyl-L-methionine analogues
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A chemoenzymatic platform for the synthesis of S-adenosyl-L-methionine (SAM) analogues compatible with downstream SAM-utilizing enzymes is reported. Forty-four non-native S/Se-alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non-native SAM analogues. As a proof of concept for the feasibility of natural product alkylrandomization , a small set of differentially-alkylated indolocarbazole analogues was generated by using a coupled hMAT2-RebM system (RebM is the sugar C4′-O-methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes. Mix and MATch: Methionine adenosyltransferase (MAT) was used to synthesize S-adenosylmethionine (SAM) analogues in a method directly compatible with downstream SAM-utilizing enzymes. As a proof of concept for the feasibility of natural product alkylrandomization by using this method, a coupled strategy in which MAT was applied in conjunction with the methyltransferase RebM was used to generate a small set of indolocarbazole analogues.
- Singh, Shanteri,Zhang, Jianjun,Huber, Tyler D.,Sunkara, Manjula,Hurley, Katherine,Goff, Randal D.,Wang, Guojun,Zhang, Wen,Liu, Chunming,Rohr, Juergen,Van Lanen, Steven G.,Morris, Andrew J.,Thorson, Jon S.
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supporting information
p. 3965 - 3969
(2014/05/06)
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- Radical SAM Activation of the B12-Independent Glycerol Dehydratase Results in Formation of 5′-Deoxy-5′-(methylthio) adenosine and Not 5′-Deoxyadenosine
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Activation of glycyl radical enzymes (GREs) by S-adenosylmethonine (AdoMet or SAM)-dependent enzymes has long been shown to proceed via the reductive cleavage of SAM. The AdoMet-dependent (or radical SAM) enzymes catalyze this reaction by using a [4Fe-4S] cluster to reductively cleave AdoMet to form a transient 5′deoxyadenosyl radical and methionine. This radical is then transferred to the GRE, and methionine and 5′deoxyadenosine are also formed. In contrast to this paradigm, we demonstrate that generation of a glycyl radical on the B12-independent glycerol dehydratase by the glycerol dehydratase activating enzyme results in formation of 5′deoxy- 5′(methylthio) adenosine and not 5′deoxyadenosine. This demonstrates for the first time that radical SAM activases are also capable of an alternative cleavage pathway for SAM.
- Demick, Jonathan M.,Lanzilotta, William N.
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body text
p. 440 - 442
(2012/03/27)
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- New insights into the design of inhibitors of human S-adenosylmethionine decarboxylase: studies of adenine C8 substitution in structural analogues of S-adenosylmethionine
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S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and depends on a pyruvoyl group for the decarboxylation process. The crystal structures of the enzyme with various inhibitors at the active site have shown that the adenine base of the ligands adopts an unusual syn conformation when bound to the enzyme. To determine whether compounds that favor the syn conformation in solution would be more potent AdoMetDC inhibitors, several series of AdoMet substrate analogues with a variety of substituents at the 8-position of adenine were synthesized and analyzed for their ability to inhibit hAdoMetDC. The biochemical analysis indicated that an 8-methyl substituent resulted in more potent inhibitors, yet most other 8-substitutions provided no benefit over the parent compound. To understand these results, we used computational modeling and X-ray crystallography to study C8-substituted adenine analogues bound in the active site.
- McCloskey, Diane E.,Bale, Shridhar,Secrist III, John A.,Tiwari, Anita,Moss III, Thomas H.,Valiyaveettil, Jacob,Brooks, Wesley H.,Guida, Wayne C.,Pegg, Anthony E.,Ealick, Steven E.
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experimental part
p. 1388 - 1407
(2009/12/07)
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- Design, synthesis, and biological evaluation of novel human 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAP) substrates
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The structure-based design, chemical synthesis, and biological evaluation of novel MTAP substrates are described. These compounds incorporate various C5′-moieties and are shown to have different kcat/Km values compared with the natural MTAP substrate (MTA).
- Kung, Pei-Pei,Zehnder, Luke R.,Meng, Jerry J.,Kupchinsky, Stanley W.,Skalitzky, Donald J.,Johnson, M. Catherine,Maegley, Karen A.,Ekker, Anne,Kuhn, Leslie A.,Rose, Peter W.,Bloom, Laura A.
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p. 2829 - 2833
(2007/10/03)
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- Adenylate Deaminase (5′-Adenylic Acid Deaminase, AMPDA)-Catalyzed Deamination of 5′-Deoxy-5′-Substituted and 5′-Protected Adenosines: A Comparison with the Catalytic Activity of Adenosine Deaminase (ADA)
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The enzyme adenylate deaminase (AMPDA) is able to catalyze the hydrolytic deamination of 5′-substituted and 5′-protected 5′ -deoxyadenosines, whereas limited or no activity is shown by adenosine deaminase (ADA) towards the same substrates. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003.
- Ciuffreda, Pierangela,Loseto, Angela,Alessandrini, Laura,Terraneo, Giancarlo,Santaniello, Enzo
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p. 4748 - 4751
(2007/10/03)
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- A CONVENIENT METHOD FOR THE SYNTHESIS AND RANEY NICKEL DESULFURIZATION OF 5'-DEOXY-5'-METHYLTHIOADENOSINE
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A convenient procedure for the preparation of 5'-deoxy-5'-methylthioadenosine is reported.Chlorination of adenosine with thionyl chloride yielded 5'-chloro-5'-deoxyadenosine.Reaction of 5'-chloro-5'deoxyadenosine with aqueous methylmercaptide anion yielded 5'-deoxy-5'-methylthioadenosine.Hydrogenolysis of 5'-deoxy-5'-methylthioadenosine over Raney nickel in water produced 5'-deoxyadenosine.This procedure affords a high yield of readily purified 5'-deoxyadenosine while avoiding the use of anhydrous solvents and pyrophoric reagents.The procedure illustrates the utility of sulfur reagents to accomplish high value added transformations in nucleoside chemistry.Key words: 5'-Deoxyadenosine; 5'-deoxy-5'methylthioadenosine; 5'-chloro-5'-deoxyadenosine; Raney nickel desulfurization.
- Scovill, John P.,Thigpen, Don L.,Lemley, Paul V.
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p. 149 - 152
(2007/10/02)
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- Synthesis and Antiproliferative Effects of Novel 5'-Fluorinated Analogues of 5'-Deoxy-5'-(methylthio)adenosine
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5'-Deoxy-5'-adenosine (9) and 5'-deoxy-5'-fluoro-5'-(methylthio)adenosine (10), two novel analogues of 5'-deoxy-5'-(methylthio)adenosine (MTA), have been synthesized and evaluated for their substrate and inhibitory activities toward MTA phosphorylase and for their biological effects in L1210 (MTA phosphorylase deficient) and L5178Y (MTA phosphorylase containing) murine leukemia cell lines.Compound 9 was a potent competitive inhibitor of MTA phosphorylase with a Ki value of 3.3 μM and was also a substrate, with activity approximately 53percent that of MTA.Compound 10 was significantly less inhibitory toward the phosphorylase with a Ki value of 141 μM; its lack of substrate activity was attributed to rapid nonenzymatic degradation.The 50percent growth inhibitory concentrations (48 h) of 9 were 300 and 200 μM in L1210 and L5178Y cells, respectively; for 10, these respective values were 2 and 0.7 μM.The initial characterization of 9 in these systems reveals that it differs from MTA by not acting as a product regulator of the polyamine biosynthetic pathway.
- Sufrin, Janice R.,Spiess, Arthur J.,Kramer, Debora L.,Libby, Paul R.,Porter, Carl W.
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p. 997 - 1001
(2007/10/02)
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- Synthesis of 5'-Alkylthio-5'-deoxynucleosides from Nucleosides in a One-pot Reaction
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5'-Alkyl(aryl)thio-5'-deoxynucleosides were prepared in high yields by the reaction of nucleosides with dialkyl or diaryl disulphides in the presence of tri-n-butylphosphine.The method is widely applicable to the synthesis of unsymmetrical sulphides.
- Nakagawa, Iwao,Aki, Koichi,Hata, Tsujiaki
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p. 1315 - 1318
(2007/10/02)
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- Methylase Models: Intramolecular Alkylation of a Phenol by an Adenosyl Sulphonium Salt
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The newly synthesized adenosylsulphonium ion, (2), undergoes a facile ring closure reaction in aqueous oxyanion buffers; in contrast, amine buffers effect an intermolecular dealkylation in competition with the lyate reaction.
- Vasquez, Peter J.,Coward, James K.
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p. 698 - 699
(2007/10/02)
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