- Structural Insights into the Mechanism of the Radical SAM Carbide Synthase NifB, a Key Nitrogenase Cofactor Maturating Enzyme
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Nitrogenase is a key player in the global nitrogen cycle, as it catalyzes the reduction of dinitrogen into ammonia. The active site of the nitrogenase MoFe protein corresponds to a [MoFe7S9C-(R)-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF (for NItrogen Fixation) assembly machinery. Among them, the radical SAM protein NifB plays an essential role, concomitantly inserting a carbide ion and coupling two [Fe4S4] clusters to form a [Fe8S9C] precursor called NifB-co. Here we report on the X-ray structure of NifB from Methanotrix thermoacetophila at 1.95 ? resolution in a state pending the binding of one [Fe4S4] cluster substrate. The overall NifB architecture indicates that this enzyme has a single SAM binding site, which at this stage is occupied by cysteine residue 62. The structure reveals a unique ligand binding mode for the K1-cluster involving cysteine residues 29 and 128 in addition to histidine 42 and glutamate 65. The latter, together with cysteine 62, belongs to a loop inserted in the active site, likely protecting the already present [Fe4S4] clusters. These two residues regulate the sequence of events, controlling SAM dual reactivity and preventing unwanted radical-based chemistry before the K2 [Fe4S4] cluster substrate is loaded into the protein. The location of the K1-cluster, too far away from the SAM binding site, supports a mechanism in which the K2-cluster is the site of methylation.
- Echavarri-Erasun, Carlos,Fajardo, Ana Sosa,Legrand, Pierre,Martin, Lydie,Nicolet, Yvain,Payá-Tormo, Luc?a,Pellicer Mart?nez, Maria Teresa,Rubio, Luis M.,Vernède, Xavier
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- Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens
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Nitrogenase biosynthesis protein NifB catalyzes the radical S-adenosyl-L-methionine (SAM)-dependent insertion of carbide into the M cluster, the cofactor of the molybdenum nitrogenase from Azotobacter vinelandii. Here, we report the identification and characterization of two naturally €truncated€ homologs of NifB from Methanosarcina acetivorans (NifBMa) and Methanobacterium thermoautotrophicum (NifBMt), which contain a SAM-binding domain at the N terminus but lack a domain toward the C terminus that shares homology with NifX, an accessory protein in M cluster biosynthesis. NifBMa and NifBMt are monomeric proteins containing a SAM-binding [Fe4S4] cluster (designated the SAM cluster) and a [Fe4S4]-like cluster pair (designated the K cluster) that can be processed into an [Fe8S9] precursor to the M cluster (designated the L cluster). Further, the K clusters in NifBMa and NifBMt can be converted to L clusters upon addition of SAM, which corresponds to their ability to heterologously donate L clusters to the biosynthetic machinery of A. vinelandii for further maturation into the M clusters. Perhaps even more excitingly, NifBMa and NifBMt can catalyze the removal of methyl group from SAM and the abstraction of hydrogen from this methyl group by 5€-deoxyadenosyl radical that initiates the radical-based incorporation of methyl-derived carbide into the M cluster. The successful identification of NifBMa and NifBMt as functional homologs of NifB not only enabled classification of a new subset of radical SAM methyltransferases that specialize in complex metallocluster assembly, but also provided a new tool for further characterization of the distinctive, NifB-catalyzed methyl transfer and conversion to an iron-bound carbide.
- Fay, Aaron W.,Wiig, Jared A.,Lee, Chi Chung,Hu, Yilin
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- Catalysis of a new ribose carbon-insertion reaction by the molybdenum cofactor biosynthetic enzyme MoaA
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MoaA, a radical S-adenosylmethionine enzyme, catalyzes the first step in molybdopterin biosynthesis. This reaction involves a complex rearrangement in which C8 of guanosine triphosphate is inserted between C2′ and C3′ of the ribose. This study identifies the site of initial hydrogen atom abstraction by the adenosyl radical and advances a mechanistic proposal for this unprecedented reaction.
- Mehta, Angad P.,Hanes, Jeremiah W.,Abdelwahed, Sameh H.,Hilmey, David G.,H?nzelmann, Petra,Begley, Tadhg P.
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- Radical S-Adenosyl Methionine Enzyme BlsE Catalyzes a Radical-Mediated 1,2-Diol Dehydration during the Biosynthesis of Blasticidin S
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The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4′-keto-3′-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 ?) and BlsE·SAM·CGA (2.62 ?) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4′ α-hydroxyalkyl radical as opposed to 1,2-migration of the C3′-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.
- Chen, Ridao,Feng, Jianqiang,Gao, Jin-Ming,Hou, Xueli,Lee, Yu-Hsuan,Liu, Hung-Wen,Liu, Xiao,Ruszczycky, Mark W.,Wang, Binju,Zhou, Jiahai
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supporting information
p. 4478 - 4486
(2022/03/18)
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- The B12-independent glycerol dehydratase activating enzyme from Clostridium butyricum cleaves SAM to produce 5′-deoxyadenosine and not 5′-deoxy-5′-(methylthio)adenosine
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Glycerol dehydratase activating enzyme (GD-AE) is a radical S-adenosyl-L-methionine (SAM) enzyme that installs a catalytically essential amino acid backbone radical onto glycerol dehydratase in bacteria under anaerobic conditions. Although GD-AE is closely homologous to other radical SAM activases that have been shown to cleave the S-C(5′) bond of SAM to produce 5′-deoxyadenosine (5’-dAdoH) and methionine, GD-AE from Clostridium butyricum has been reported to instead cleave the S-C(γ) bond of SAM to yield 5′-deoxy-5′-(methylthio)adenosine (MTA). Here we re-investigate the SAM cleavage reaction catalyzed by GD-AE and show that it produces the widely observed 5’-dAdoH, and not the less conventional product MTA.
- Walls, William G.,Moody, James D.,McDaniel, Elizabeth C.,Villanueva, Maria,Shepard, Eric M.,Broderick, William E.,Broderick, Joan B.
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- HygY Is a Twitch Radical SAM Epimerase with Latent Dehydrogenase Activity Revealed upon Mutation of a Single Cysteine Residue
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HygY is a SPASM/twitch radical SAM enzyme hypothesized to catalyze the C2′-epimerization of galacamine during the biosynthesis of hygromycin B. This activity is confirmed via biochemical and structural analysis of the derivatized reaction products using chemically synthesized deuterated substrate, high-resolution mass spectrometry and1H NMR. Electron paramagnetic resonance spectroscopy of the reduced enzyme is consistent with ligation of two [Fe4S4] clusters characteristic of the twitch radical SAM subgroup. HygY catalyzed epimerization proceeds with incorporation of a single solvent Hydron into the talamine product facilitated by the catalytic cysteine-183 residue. Mutation of this cysteine to alanine converts HygY from a C2′-epimerase to an C2′-dehydrogenase with comparable activity. The SPASM/twitch radical SAM enzymes often serve as anaerobic oxidases making the redox-neutral epimerases in this class rather interesting. The discovery of latent dehydrogenase activity in a twitch epimerase may therefore offer new insights into the mechanistic features that distinguish oxidative versus redox-neutral SPASM/twitch enzymes and lead to the evolution of new enzyme activities.
- Besandre, Ronald A.,Chen, Zhang,Davis, Ian,Liu, Aimin,Liu, Hung-Wen,Ruszczycky, Mark Walter,Zhang, Jiawei
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supporting information
p. 15152 - 15158
(2021/09/29)
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- Reductive Cleavage of Sulfoxide and Sulfone by Two Radical S-Adenosyl- l -methionine Enzymes
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Sulfoxides and sulfones are commonly found in nature as a result of thioether oxidation, whereas only a very few enzymes have been found to metabolize these compounds. Utilizing the strong reduction potential of the [4Fe-4S] cluster of radical S-adenosyl-l-methionine (SAM) enzymes, we herein report the first enzyme-catalyzed reductive cleavage of sulfoxide and sulfone. We show two radical SAM enzymes, tryptophan lyase NosL and the class C radical SAM methyltransferase NosN, are able to act on a sulfoxide SAHO and a sulfone SAHO2, both of which are structurally similar to SAM. NosL cleaves all of the three bonds (i.e., S-C(5′), S-C(γ), and S-O) connecting the sulfur center of SAHO, with a preference for S-C(5′) bond cleavage. Similar S-C cleavage activity was also found for SHAO2, but no S-O cleavage was observed. In contrast to NosL, NosN almost exclusively cleaves the S-C(5′) bonds of SAHO and SAHO2 with much higher efficiencies. Our study provides valuable insights into the [4Fe-4S] cluster-mediated reduction reactions and highlights the remarkable catalytic promiscuity of radical SAM enzymes.
- Mandalapu, Dhanaraju,Ji, Xinjian,Zhang, Qi
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- Mechanistic Investigations of PoyD, a Radical S-Adenosyl- l -methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis
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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of bioactive peptides. Among RiPPs, the bacterial toxin polytheonamide A is characterized by a unique set of post-translational modifications catalyzed by novel radical S-adenosyl-l-methionine (SAM) enzymes. Here we show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide epimerization in a C-to-N directional manner. By combining mutagenesis experiments with labeling studies and investigating the enzyme substrate promiscuity, we deciphered in detail the mechanism of PoyD. We notably identified a critical cysteine residue as a likely key H atom donor and demonstrated that PoyD belongs to a distinct family of radical SAM peptidyl epimerases. In addition, our study shows that the core peptide directly influences the epimerization pattern allowing for production of peptides with unnatural epimerization patterns.
- Parent, Aubérie,Benjdia, Alhosna,Guillot, Alain,Kubiak, Xavier,Balty, Clémence,Lefranc, Benjamin,Leprince, Jér?me,Berteau, Olivier
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p. 2469 - 2477
(2018/02/28)
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- Mechanism of a Class C Radical S-Adenosyl- l -methionine Thiazole Methyl Transferase
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The past decade has seen the discovery of four different classes of radical S-adenosylmethionine (rSAM) methyltransferases that methylate unactivated carbon centers. Whereas the mechanism of class A is well understood, the molecular details of methylation by classes B-D are not. In this study, we present detailed mechanistic investigations of the class C rSAM methyltransferase TbtI involved in the biosynthesis of the potent thiopeptide antibiotic thiomuracin. TbtI C-methylates a Cys-derived thiazole during posttranslational maturation. Product analysis demonstrates that two SAM molecules are required for methylation and that one SAM (SAM1) is converted to 5′-deoxyadenosine and the second SAM (SAM2) is converted to S-adenosyl-l-homocysteine (SAH). Isotope labeling studies show that a hydrogen is transferred from the methyl group of SAM2 to the 5′-deoxyadenosine of SAM1 and the other two hydrogens of the methyl group of SAM2 appear in the methylated product. In addition, a hydrogen appears to be transferred from the β-position of the thiazole to the methyl group in the product. We also show that the methyl protons in the product can exchange with solvent. A mechanism consistent with these observations is presented that differs from other characterized radical SAM methyltransferases.
- Zhang, Zhengan,Mahanta, Nilkamal,Hudson, Graham A.,Mitchell, Douglas A.,Van Der Donk, Wilfred A.
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p. 18623 - 18631
(2017/12/26)
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- Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme
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Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na+ as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M+ ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[13C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li+ to Cs+, PFL-AE activity sharply maximizes at K+, with NH4+ closely matching the efficacy of K+. PFL-AE is thus a type I M+-activated enzyme whose M+ controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.
- Shisler, Krista A.,Hutcheson, Rachel U.,Horitani, Masaki,Duschene, Kaitlin S.,Crain, Adam V.,Byer, Amanda S.,Shepard, Eric M.,Rasmussen, Ashley,Yang, Jian,Broderick, William E.,Vey, Jessica L.,Drennan, Catherine L.,Hoffman, Brian M.,Broderick, Joan B.
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p. 11803 - 11813
(2017/09/08)
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- Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase
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RimO is a member of the growing radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a reduced [4Fe-4S] cluster to effect reductive cleavage of the 5′ C-S bond of SAM to form a 5′-deoxyadenosyl 5′-radical (5′-dA? intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (-SCH3) to C3 of aspartate 89 of protein S12, one of 21 proteins that compose the 30S subunit of the bacterial ribosome. However, the exact mechanism by which this transformation takes place has remained elusive. Herein, we describe the stereochemical course of the RimO reaction. Using peptide mimics of the S12 protein bearing deuterium at the 3 pro-R or 3 pro-S positions of the target aspartyl residue, we show that RimO from Bacteroides thetaiotaomicron (Bt) catalyzes abstraction of the pro-S hydrogen atom, as evidenced by the transfer of deuterium into 5′-deoxyadenosine (5′-dAH). The observed kinetic isotope effect on H atom versus D atom abstraction is ~1.9, suggesting that this step is at least partially rate determining. We also demonstrate that Bt RimO can utilize the flavodoxin/flavodoxin oxidoreductase/NADPH reducing system from Escherichia coli as a source of requisite electrons. Use of this in vivo reducing system decreases, but does not eliminate, formation of 5′-dAH in excess of methylthiolated product.
- Landgraf, Bradley J.,Booker, Squire J.
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supporting information
p. 2889 - 2892
(2016/03/19)
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- Anaerobic 5-Hydroxybenzimidazole Formation from Aminoimidazole Ribotide: An Unanticipated Intersection of Thiamin and Vitamin B12 Biosynthesis
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Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralogue of thiC (bzaF) clustered with anaerobic vitamin B12 biosynthetic genes. Here we demonstrate that BzaF is a radical S-adenosylmethionine enzyme that catalyzes the remarkable conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI). We identify the origin of key product atoms and propose a reaction mechanism. These studies represent the first step in solving a long-standing problem in anaerobic vitamin B12 assembly and reveal an unanticipated intersection of thiamin and vitamin B12 biosynthesis.
- Mehta, Angad P.,Abdelwahed, Sameh H.,Fenwick, Michael K.,Hazra, Amrita B.,Taga, Michiko E.,Zhang, Yang,Ealick, Steven E.,Begley, Tadhg P.
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supporting information
p. 10444 - 10447
(2015/09/28)
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- Delineating the biosynthesis of gentamicin X2, the common precursor of the gentamicin C antibiotic complex
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Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.
- Huang, Chuan,Huang, Fanglu,Moison, Eileen,Guo, Junhong,Jian, Xinyun,Duan, Xiaobo,Deng, Zixin,Leadlay, Peter F.,Sun, Yuhui
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p. 251 - 261
(2015/07/15)
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- In vitro reconstitution of the radical S-adenosylmethionine enzyme MqnC involved in the biosynthesis of futalosine-derived menaquinone
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The radical S-adenosylmethionine enzyme MqnC catalyzes conversion of dehypoxanthine futalosine (DHFL) to the unique spiro compound cyclic DHFL in the futalosine pathway for menaquinone biosynthesis. This study describes the in vitro reconstitution of [4Fe-4S] cluster-dependent MqnC activity and identifies the site of abstraction of a hydrogen atom from DHFL by the adenosyl radical.
- Cooper, Lisa E.,Fedoseyenko, Dmytro,Abdelwahed, Sameh H.,Kim, Soong-Hyun,Dairi, Tohru,Begley, Tadhg P.
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p. 4592 - 4594
(2013/07/26)
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- Biosynthesis of F0, precursor of the F420 cofactor, requires a unique two radical-SAM domain enzyme and tyrosine as substrate
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Cofactors play key roles in metabolic pathways. Among them F420 has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F0-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. We report here the purification of both types of F 0-synthase and their in vitro activities. Our study allows us to establish that F0-synthase, from both types, uses 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, our data support the fact that F0-synthase generates two 5′- deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes.
- Decamps, Laure,Berteau, Olivier,Philmus, Benjamin,Begley, Tadhg P.,Benjdia, Alhosna,White, Robert
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supporting information
p. 18173 - 18176,4
(2012/12/12)
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- Biosynthesis of F0, precursor of the F420 cofactor, requires a unique two radical-SAM domain enzyme and tyrosine as substrate
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Cofactors play key roles in metabolic pathways. Among them F420 has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F0-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. We report here the purification of both types of F 0-synthase and their in vitro activities. Our study allows us to establish that F0-synthase, from both types, uses 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, our data support the fact that F0-synthase generates two 5′- deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes.
- Decamps, Laure,Philmus, Benjamin,Benjdia, Alhosna,White, Robert,Begley, Tadhg P.,Berteau, Olivier
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supporting information
p. 18173 - 18176
(2013/01/15)
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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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scheme or table
p. 440 - 442
(2012/03/27)
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- A Radical dance in thiamin biosynthesis: Mechanistic analysis of the bacterial hydroxymethylpyrimidine phosphate synthase
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Tricky things with ThiC: Hydroxymethylpyrimidine phosphate (HMP-P) synthase (ThiC) catalyzes one of the most complex rearrangement reactions in primary metabolism. Deuteration experiments show that under reducing conditions, in the presence of aminoimidazole ribonucleotide, the 5-deoxyadenosyl radical generated at the active site of ThiC reacts directly with the substrate and performs two iterative hydrogen atom abstraction events to catalyze this rearrangement (see scheme; SAM=S-adenosylmethionine).
- Chatterjee, Abhishek,Hazra, Amrita B.,Abdelwahed, Sameh,Hilmey, David G.,Begley, Tadhg P.
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supporting information; experimental part
p. 8653 - 8656
(2011/01/06)
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- Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli
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Thiazole synthase in Escherichia coli is an αβ heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the Cα-Cβ bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S]+ cluster to reductively cleave AdoMet, forming methionine and a 5′-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5′- deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (Cα-Cβ bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle.
- Challand, Martin R.,Martins, Filipa T.,Roach, Peter L.
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experimental part
p. 5240 - 5248
(2011/02/25)
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- [FeFe]-Hydrogenase Cyanide Ligands Derived from S-Adenosylmethionine- Dependent Cleavage of Tyrosine
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"Chemical Equation Presented" What's your poison? Hydrogenases catalyze the reversible formation of dihydrogen from two electrons and two protons. The maturation of the [FeFe]-hydrogenase active-site cofactor (H cluster) requires three gene products, HydE
- Driesener, Rebecca C.,Challand, Martin R.,McGlynn, Shawn E.,Shepard, Eric M.,Boyd, Eric S.,Broderick, Joan B.,Peters, John W.,Roach, Peter L.
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supporting information; experimental part
p. 1687 - 1690
(2010/06/16)
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- Mechanistic investigations of anaerobic sulfatase-maturating enzyme: Direct Cβ H-atom abstraction catalyzed by a radical AdoMet enzyme
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(Chemical Equation Presented) Sulfatases are unique in requiring an essential post-translational modification of a critical active-site cysteinyl or seryl residue to 3-oxoalanine usually called Cα-formylglycine(FGly). This post-translational modification is catalyzed anaerobically by anaerobic Sulfatase Maturating Enzyme (anSME), a member of the radic al AdoMet superfamily. Using a new labeled substrate, we demonstrate that anSME uses a 5′-deoxyadenosyl radical to catalyze direct H-atom abstraction from the substrate. We thus established that anSMEs are the first radical AdoMet enzymes catalyzing a post-translational modification involving Cβ H-atom abstraction from an active site cysteinyl or seryl residue. This mechanistic study allowed us to decipher the first steps of the mechanism of this new radical AdoMet enzyme family.
- Benjdia, Alhosna,Leprince, Jerome,Sandstroem, Corine,Vaudry, Hubert,Berteau, Olivier
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supporting information; experimental part
p. 8348 - 8349
(2009/10/23)
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- Radical reactions in aqueous medium using (Me3Si)3SiH
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(Chemical Equation Presented) (Me3Si)3SiH was used as a successful reagent in a variety of radical-based transformations in water. The system comprising substrate, silane, and initiator (ACCN) mixed in aqueous medium at 100°C worked well for both hydrophilic and hydrophobic substrates, with the only variation that an amphiphilic thiol was also needed in case of the water-soluble compounds.
- Postigo, Al,Kopsov, Sergey,Ferreri, Carla,Chatgilialoglu, Chryssostomos
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p. 5159 - 5162
(2008/09/17)
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- The nucleoside transport proteins, NupC and NupG, from Escherichia coli: Specific structural motifs necessary for the binding of ligands
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A series of 46 natural nucleosides and analogues (mainly adenosine-based) were tested as inhibitors of [U-14C]uridine uptake by the concentrative, H+-linked nucleoside transport proteins NupC and NupG from Escherichia coli. The two evolutionarily unrelated transporters showed similar but distinct patterns of inhibition, revealing differing selectivities for the different nucleosides and their analogues. Binding of nucleosides to NupG required the presence of hydroxyl groups at each of the C-3′ and C-5′ positions of ribose, while binding to NupC required only the C-3′ hydroxyl substituent. The greater importance of the ribose moiety for binding to NupG is consistent with the evolutionary relationship between this protein and the oligosaccharide: H+ symporter (OHS) subfamily of the major facilitator superfamily (MFS) of transporters. For both proteins the natural α-configuration at C-3′ and the natural β-configuration at C-1′ was mandatory for ligand binding. N-7 in the imidazole ring of adenosine and the amino group at C-6 were found not to be important for binding and both transporters showed flexibility for substitution at C-6/N6; one or both of N-l and N-3 were important for adenosine analogue binding to NupC but significantly less so for binding to NupG. From the different effects of 8-bromoadenosine on the two transporters it appears that adenosine selectively binds to NupC in an anti- rather than a syn-conformation, whereas NupG is less prescriptive. The pattern of inhibition of NupC by differing nucleoside analogues confirmed the functional relationship of the bacterial transporter to members of the human concentrative nucleoside transporter (CNT) family and reaffirmed the use of the bacterial protein as an experimental model for these physiologically and clinically important mammalian proteins. The specificity data for NupG have been used to develop a homology model of the protein's binding site, based on the X-ray crystallographic structure of the disaccharide transporter LacY from E. coli. We have also developed an efficient general protocol for the synthesis of adenosine and three of its analogues, which is illustrated by the synthesis of [1′-13C]adenosine.
- Patching, Simon G.,Baldwin, Stephen A.,Baldwin, Alexander D.,Young, James D.,Gallagher, Maurice P.,Henderson, Peter J. F.,Herbert, Richard B.
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p. 462 - 470
(2007/10/03)
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- Preparation of deoxynucleosides
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Methods for preparing deoxynucleosides from their corresponding ribonucleosides by forming 3-tert-butylphenoxythiocarbonylderivatives of the ribonucleosides and subsequently effecting radical deoxygenation reactions at the carbon atoms to be deoxygenated.
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Page column 17
(2008/06/13)
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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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- REACTION OF ADENINE NUCLEOSIDES, TOSYLATED IN THE CARBOHYDRATE MOIETY, WITH LITHIUM TRIETHYLBOROHYDRIDE
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The reaction of lithium triethylborohydride with the 2',3'-di-O-p-tolylsulphonyl derivatives of 9-β-D-ribofuranosyladenine, 9-β-D-arabinofuranosyladenine, 9-β-D-xylofuranosyladenine and 9-β-D-lyxofuranosyladenine was studied.The reaction of 2',3'-di-O-p-tolylsulphonyladenosine with LiEt3BH gave 9-(3-deoxy-β-D-threo-pentofuranosyl)adenine.This rearrangement reaction was used for the synthesis of 9-(3,5-dideoxy-β-D-threo-pentofuranosyl)adenine in one step from 2',3',5'-tri-O-p-tolylsulphonyladenosine in 58percent yield.The p-tolylsulphonyl group in the 2'-"up" configuration of unprotected adenine nucleosides was preferentially attacked by LiEt3BH giving S-O-bond scission.This was shown by the formation of 9-(3-deoxy-β-D-threo-pentofuranosyl)adenine from 2',3'-di-O-p-tolylsulphonyl-9-β-D-arabinofuranosyladenine and by the formation of 9-β-D-lyxofuranosyladenine from 2'-O-p-tolylsulphonyl-9-β-D-lyxofuranosyladenine with LiEt3BH. 9-β-D-Lyxofuranosyladenine was synthesized from 3',5'-di-O-benzoyl-9-β-D-xylofuranosyladenosine in 88percent yield using a triflate displacement reaction.
- Herdewijn, Piet
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p. 6563 - 6580
(2007/10/02)
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- Thermolysis of the Co-O Bond of Adenosylcobalamin. 2. Products, Kinetics, and Co-O Bond Dissociatiom Energy in Aqueous Solution
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The reaction product, kinetic, ΔHexct., ΗSexct., and Co-O bond dissociation energies (BDEs) are reported for the anaerobic, thermal decomposition of adenosylcobalamin (AdoB12) in aqueous solution.These studies reveal that the reaction proceeds via two competing pathways, heterolytic Co-O bond clevage to yield aquocobalamin, adenine, and a sugar residue and competing Co-O bond homolysis to give CoIIB12 and 8,5'-anhydroadenosine.At pH 4.0, heterolysis is the major mode (88percent at 85 deg C) of decomposition, while at pH 7.0 homolysis dominates (90percent at 85 degC).The temperature dependence of the rate of AdoB12 Co-O bond homolysis in neutral H2O was obtained from 85.0 to 110.0 deg C, yielding ΔHexct.h=31.8 +/- 0.7 kcal/mol and ΔSexct.h=6.8 +/- 1.0 eu.These values are significantly different from previously reported aqueous (pH 4.3) values of ΔHexct.h=26.3 +/- 0.6 kcal/mol and ΔSexct.h=-6 +/- 2 eu.The temperature dependence of the axial base equilibrium of AdoB12 in neutral water was measured, yielding ΔH=-5.6 +/- 0.9 kcal/mol and ΔS=-13 +/- 3 eu.Combining the above results with other data yields an estimate of 30 +/- 2 kcal/mol for the base-on Co-O BDE of AdoB12 in water, in good agreement with our previously reported value of 31.5 +/- 1.3 kcal/mol obtained in ethylene glycol.
- Hay, Benjamin P.,Finke, Richard G.
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p. 4820 - 4829
(2007/10/02)
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- REACTION OF NUCLEOSIDES WITH THIONYL CHLORIDE; PREPARATION OF THE DEOXY DERIVATIVES OF CYTIDINE AND ADENOSINE
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On reaction of thionyl chloride with cytidine and adenosine in refluxing acetonitrile, the 5'-chloro-2',3'-sulphinyl derivatives I and VII are formed in a quantitative yield.On heating in dimethylformamide, compound I affords 5'-chloro-5'-deoxycyclocytidine (II) which is hydrolyzed in alkali to the arabinosyl derivative III; reduction of III with tributyltin hydride gives the 5'-deoxyarabinosyl derivative IV.The sulphinyl derivative I is hydrolyzed to 5'-chloro-5'-deoxycytidine (V) which is reduced to 5'-deoxycytidine (VI).Analogously, the sulphinyl derivative VII affords 5'-chloro-5'-deoxyadenosine (VIII) and the reduction of VIII gives 5'-deoxyadenosine (IX).Of these compounds, the 5'-chloro-5'-deoxyarabinosyl derivative as the only one shows an inhibitory effect towards the L1210 cell growth.
- Hrebabecky, Hubert,Brokes, Josef,Beranek, Jiri
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p. 599 - 605
(2007/10/02)
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