18O Studies of the 2-Ketoglutarate-dependent Sequential Oxygenation of Thymine to 5-Carboxyuracil

Holme, Elisabeth; Lindstedt, Göran; Lindstedt, Sven; Tofft, Marianne

doi:10.1016/s0021-9258(18)62229-x

Cited by 41 publications

(18 citation statements)

References 21 publications

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“…One of the first Fe II /aKG-dependent dioxygenases to be discovered was thymine 7-hydroxylase (T7H), an enzyme that catalyzes three consecutive oxidations of thymine to form 5-hydroxymethyl uracil (HMU), 5-formyl uracil (FU), and uracil-carboxylic acid (Scheme 2). [13][14][15][16] Each of these reactions requires Fe II , aKG, and O 2 and produces stoichiometric amounts of the respective primary product, CO 2 , and succinate. One atom of molecular oxygen is incorporated into succinate and the other into the primary products.…”

Section: Thymine 7-hydroxylasementioning

confidence: 99%

“…One atom of molecular oxygen is incorporated into succinate and the other into the primary products. [15][16][17] The R. glutinis T7H sequence includes His 211, Asp 213, and His 268 as putative metal-binding ligands and Arg 284 as a likely aKG stabilizing residue, but no structural studies have been carried out to confirm these assignments.…”

Section: Thymine 7-hydroxylasementioning

confidence: 99%

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FeII/α-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism

2008

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Fe II /α-ketoglutarate-dependent hydroxylases uniformly possess a double-stranded β-helix fold with two conserved histidines and one carboxylate coordinating their mononuclear ferrous ions. Oxidative decomposition of the α-keto acid is proposed to generate a ferryl-oxo intermediate capable of hydroxylating unactivated carbon atoms in a myriad of substrates. This perspective focuses on a subgroup of these enzymes that are involved in pyrimidine salvage, purine decomposition, nucleoside and nucleotide hydroxylation, DNA/RNA repair, and chromatin modification. The varied reaction schemes are presented, and selected structural and kinetic information is summarized. IntroductionFerrous ion and α-ketoglutarate (αKG)-dependent dioxygenases comprise an enzyme superfamily with members present in animals, plants, protists, fungi, bacteria and even viruses. Most representatives of this large group of enzymes couple the oxidative decarboxylation of αKG to various hydroxylation reactions ( Fig. 1), but others are capable of catalyzing desaturation, epoxidation, halogenation, ring formation, or ring expansion reactions, which allows them to fill a wide variety of biological roles including antibiotic biosynthesis, hypoxic sensing, DNA repair, and various types of metabolite transformations. [1][2][3] Not surprisingly, the primary substrates also are diverse and include proteins, lipids, nucleic acids, and a myriad of small molecules that can be used for synthesis or undergo degradation.The sequences of Fe II /αKG dioxygenases show little overall identity, yet the diverse reactions all are catalyzed by proteins with the same core fold and use similar chemistries. 4 The hallmark of these enzymes is their use of Fe II to activate molecular oxygen according to a mechanism (Scheme 1) first proposed by Hanauske-Abel and Günzler in 1982 for prolyl 4-hydroxylase. 5 (A) In the absence of substrates, a "facial triad" (usually two His plus one Asp or Glu occurring in a His-X-Asp/Glu-X n -His motif) weakly bind one face of the hexacoordinate metal. 6,7 (B) The αKG co-substrate displaces two waters and coordinates Fe II in a bidentate fashion with its C1 carboxyl and C2 keto oxygens. The binding of αKG is Correspondence to: Robert P. Hausinger, hausinge@msu.edu. NIH Public Access Author ManuscriptDalton Trans. Author manuscript; available in PMC 2010 July 20. This perspective focuses on the modification of nucleobases, nucleosides, nucleotides, and chromatin by the Fe II and αKG dependent hydroxylases (Table 1). We discuss the degradation and salvage of free bases by the fungal enzymes thymine 7-hydroxylase and xanthine hydroxylase. Next, nucleoside hydroxylases are briefly described. We then examine the developmental modification of DNA in kinetoplastid protozoans and summarize evidence that the JBP1 and JBP2 proteins possess thymidine hydroxylase activity. The oxidative repair of DNA by Escherichia coli AlkB is discussed, and the properties of mammalian homologues are summarized. Finally, we present emerging evidence pointing to...

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Section: Thymine 7-hydroxylasementioning

confidence: 99%

Section: Thymine 7-hydroxylasementioning

confidence: 99%

FeII/α-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism

2008

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“…Thymine hydroxylase (EC 1.14.11.6) catalyzes the three oxidation reactions shown in eq 1 (Holme et al, 1970(Holme et al, ,1971Liu et al, 1973). These oxidations are not processive, with Thornburg et al et al, 1971).…”

mentioning

confidence: 99%

A non-heme iron protein with heme tendencies: An investigation of the substrate specificity of thymine hydroxylase

Thornburg

Lai²,

Wishnok³

et al. 1993

Biochemistry

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Thymine hydroxylase from Rhodotorula glutinis catalyzes the oxidation of thymine to its alcohol, aldehyde, and carboxylic acid in three successive reactions. Each step involves stoichiometric consumption of O2 and alpha-ketoglutarate and formation of CO2 and succinate. Given the promiscuity of this enzyme, it was hoped that it would serve as a prototype for understanding the mechanism of this class of enzymes, the non-heme Fe2+ dioxygenases. Kinetic parameters for thymine, O2, Fe2+, and alpha-ketoglutarate have been determined, and isotope effect analysis of (trideuteriomethyl)thymine with enzyme reveals D(V) = 2.08 and D(V/K) = 1.11 at saturating O2. The kinetic parameters for (hydroxymethyl)uracil oxidation have been determined, and incubation of (5'-R)- and (5'-S)-[5'-2H]-5-(hydroxymethyl)uracil with enzyme reveals stereospecific removal of the pro-S hydrogen. No apparent isotope effect is observed in this reaction. The substrate specificity of this enzyme has been examined in detail. The enzyme can catalyze epoxidation, oxidation of a thioether to a sulfoxide and a sulfone, hydroxylation of an unactivated carbon-hydrogen bond, and oxidation of a methylamine to formaldehyde, as revealed through studies with 5-vinyluracil, 5-(methylthio)uracil, 5,6-dihydrothymine, and 1-methylthymine, respectively. In each case, the products were identified by gas chromatography-mass spectrometry, and 18O2-labeling studies revealed that one atom from O2 is incorporated into each product. The enzyme has also been shown to catalyze an uncoupling of hydroxylation and decarboxylation in the presence of a substrate analog incapable of undergoing hydroxylation or a substrate that is difficult to oxidize.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Des solutions des acides 1, 5, 6, 19, et .20 dans le methanol ont été agitées pendant 24 heures et analysées aussi bien après élimination du solvant qu'après traitement avec du diazométhane.Avec tous les acides excepté l'acide l,nous avons obtenu des quantités importantes des diméthylacétals correspondants (équ.9,tableau 11). %ç (b) 30 (37) 70 (38) 80 (39) 90 ( 33) 100 ( 40)…”

Section: Discussionunclassified

“…A partir de 1.5 gr (10 mmoles) d'acide phénylglyoxalique dans 100 ml de methanol,on obtient après traitement avec du diazométhane,1.96 gr d'un mélange constitué de 70% de diméthoxy-2,2 phénylacétate de méthyle (4_1) et de 30% de phénylglyoxalate de méthyle ( 37 A partir de 1.3 gr (10 mmoles) d'acide diméthyl-3,3 oxo-2 butyrique dans 100 ml de methanol,on obtient après traitement avec du diazométhane,1.58 gr de mélange constitué de 30% de ^2 et de 70% de diméthyl-3,3 oxo-2 butyrate de méthyle (38) A partir de 0.88 gr d'acide pyruvique dans 100 ml de methanol et après traitement avec du diazométhane,on obtient 1.07 gr de mélange constitué de 10 % de diméthoxy-2,2 propionate de méthyle (4_4) identique au produit synthétisé par la méthode de Conia'…”

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Decarboxylation Oxydative Des Acides Α-Cetoniques. Modeles Pour l'HYDROXYLATIONBIOLOGIQUE

Αγορίτσα-Εξάρχου¹

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18O Studies of the 2-Ketoglutarate-dependent Sequential Oxygenation of Thymine to 5-Carboxyuracil

Cited by 41 publications

References 21 publications

FeII/α-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism

FeII/α-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism

A non-heme iron protein with heme tendencies: An investigation of the substrate specificity of thymine hydroxylase

Decarboxylation Oxydative Des Acides Α-Cetoniques. Modeles Pour l'HYDROXYLATIONBIOLOGIQUE

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