7-Hydroxylation of thymine in a neurospora strain coupled to oxidative decarboxylation of 2-ketoglutarate

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

doi:10.1016/0005-2744(70)90177-4

Cited by 35 publications

(16 citation statements)

References 14 publications

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“…However, thymine hydroxylase enzymes have been characterized in fungi including Neurospora crassa and Rhodotorula glutinis (17–21). These thymine 7-hydroxylase enzymes are involved in the thymine salvage pathway catalyzing the oxygenation of thymine to 5-hydroxymethyluracil coupled to oxidative decarboxylation of 2-oxoglutarate (20). Once thymine is converted to HOMedU, it can then be converted into uracil through a series of enzymatic steps, allowing the use of thymine as a pyrimidine source in the absence of a de novo synthesis pathway.…”

Section: Discussionmentioning

confidence: 99%

JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes

Cliffe

Kieft

Southern

et al. 2009

Nucleic Acids Research

106

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Genomic DNA of African trypanosomes contains a hypermodified thymidine residue termed base J (β-d-glucosyl-HOMedU). This modified base is localized primarily to repetitive DNA, namely the telomeres, and is implicated in the regulation of antigenic variation. The base is synthesized in a two-step pathway. Initially, a thymidine residue in DNA is hydroxylated by a thymidine hydroxylase (TH). This intermediate (HOMedU) is then glucosylated to form base J. Two proteins involved in J synthesis, JBP1 (J binding protein 1) and JBP2, contain a putative TH domain related to the family of Fe2+/2-oxoglutarate-dependent hydroxylases. We have previously shown that mutations in the TH domain of JBP1 kill its ability to stimulate J synthesis. Here we show that mutation of key residues in the TH domain of JBP2 ablate its ability to induce de novo J synthesis. While the individual JBP1 null and JBP2 null trypanosomes have reduced J levels, the deletion of both JBP1 and JBP2 generates a cell line that completely lacks base J but still contains glucosyl-transferase activity. Reintroduction of JBP2 in the J-null trypanosome stimulates HOMedU formation and site-specific synthesis of base J. We conclude that JBP2 and JBP1 are the TH enzymes involved in J biosynthesis.

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Section: Discussionmentioning

confidence: 99%

JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes

Cliffe

Kieft

Southern

et al. 2009

Nucleic Acids Research

106

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“…[13][14][15][16] Each of these reactions requires Fe II , αKG, 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: Pyrimidine and Purine Hydroxylases 21 Thymine 7-hydroxylasementioning

confidence: 99%

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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“…Thymine 7-hydroxylase from the fungus Neurospora crassa was among the first 2OG-dependent oxygenases to be characterized. 259,260 The enzyme catalyses sequential oxygen additions to the methyl group of the free base, forming 5-hydroxymethyluracil, 5-formyluracil and 5-carboxyuracil ( Figure 1.11A). 261,262 Additional studies have focused on the purified fungal enzyme from Rhodotorula glutinis, 263 including analysis of its broad substrate specificity.…”

Section: Hydroxylases Of Nucleobases and Nucleosidesmentioning

confidence: 99%

Biochemical Diversity of 2-Oxoglutarate-Dependent Oxygenases

Hausinger

2015

2-Oxoglutarate-Dependent Oxygenases

119

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This chapter summarizes the diverse array of biochemical transformations that are catalysed by Fe(ii)- and 2-oxoglutarate (2OG)-dependent oxygenases. One group of these enzymes utilizes protein substrates and functions in structural stabilization, oxygen sensing, histone-dependent regulation, or other roles. A second set of 2OG-dependent oxygenases acts on polynucleotides with functions that include DNA/RNA repair, regulation of transcription, biosynthesis of unique bases, and demethylation of 5-methylcytosine. A third assemblage of enzymes in this family is involved in lipid-related metabolism and function in carnitine biosynthesis, degradation of phytanic acids, and modification of various lipids. A fourth collection of these oxygenases catalyses reactions related to synthesis of flavonoids, anthocyanins, gibberellins, alkaloids and other metabolites found predominantly in plants. A fifth group of these enzymes acts on a variety of small molecules including free amino acids, nucleobases/nucleosides, herbicides, sulfonates/sulfates and phosphonates. A sixth compilation of 2OG-dependent oxygenases is utilized for antibiotic biosynthesis, including several halogenating enzymes. Finally, a seventh set of these enzymes is related in structure or mechanism to the 2OG-dependent oxygenases, but do not utilize 2OG, and include isopenicillin N synthase, a plant-specific ethylene-forming enzyme, and two enzymes that use 4-hydroxyphenylpyruvate. This introduction to the biochemical diversity of these amazing enzymes provides a foundation for appreciating the specific aspects detailed in the remaining chapters of this text.

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7-Hydroxylation of thymine in a neurospora strain coupled to oxidative decarboxylation of 2-ketoglutarate

Cited by 35 publications

References 14 publications

JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes

JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes

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

Biochemical Diversity of 2-Oxoglutarate-Dependent Oxygenases

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