Thymine 7-hydroxylase and pyrimidine deoxyribonucleoside 2' -hydroxylase activities in Rhodotorula glutinis.

Wondrack, L M; Hsu, Chin-An; Abbott, Mitchel T.

doi:10.1016/s0021-9258(19)46961-5

Cited by 29 publications

(7 citation statements)

References 10 publications

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“…To use this nonradioactive procedure to follow enzyme activity during isolation from the host organism that contained endogenous thymine and HmU, we used deuterium-enriched thymine as the substrate. Substrate requirements and the apparent molecular weight of the isolated enzyme were consistent with previously published accounts , .…”

Section: Resultssupporting

confidence: 89%

“…The DNA sequence reported here predicted a protein of 332 amino acids, consistent with Smiley et al (17), and a predicted molecular mass of 37 kDa, consistent with the value reported by Stubbe and co-workers (14) for the wild-type enzyme. Substrate requirements and pH optimum for the cloned enzyme were indistinguishable from the isolated enzyme (11,14). In accord with Smiley et al (17), the DNA sequence predicted valine at position 9, which differs from Edman sequencing data from Stubbe and co-workers (14), who observed proline at this position.…”

Section: Th From R Glutinis Was Isolated and Examined Bysupporting

confidence: 51%

“…In addition to studies of TH isolated from N. crassa , TH isolated from Aspergillus nidulans , and Rhodotorula glutinis − has been characterized. In 1993, Stubbe and co-workers reported on studies of a partially purified fraction from the red mold R. glutinis , .…”

Section: Introductionmentioning

confidence: 99%

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Cloning and Characterization of Rhodotorula glutinis Thymine Hydroxylase

Neidigh

Darwanto

Williams

et al. 2009

Chem. Res. Toxicol.

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Thymine hydroxylase (TH) is a member of the alpha-ketoglutarate-dependent nonheme iron dioxygenase family that includes a series of DNA repair proteins including alkB. Substantial interest in this family of enzymes derives from their capacity to modify DNA bases and precursors by oxidation. Previously, a sequence has been published for cloned Rhodotorula glutinis TH. However, the minimal reported activity of this enzyme, coupled with inconsistencies with previously published mass spectrometry data, compelled us to reexamine TH. The sequence reported here differs from the previously reported sequence at two amino acid positions and is consistent with previously reported mass spectrometry data. The cloned enzyme characterized in this report displayed substantial activity, indicating that the sequence differences are critical for activity. The substrate selectivity of TH against a series of pyrimidine analogues is consistent with that reported for the wild-type enzyme and, in part, explains the mode of selection of uracil analogues. A preliminary model of the active site has been constructed for the purposes of comparing TH with other members of this family. TH and alkB share in common the capacity to oxidize N-methyl groups. However, TH has the added capacity to oxidize the 5-methyl group of thymine, a property that is potentially important for enzymes that could act on DNA and modify DNA-protein interactions.

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Section: Resultssupporting

confidence: 89%

Section: Th From R Glutinis Was Isolated and Examined Bysupporting

confidence: 51%

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Cloning and Characterization of Rhodotorula glutinis Thymine Hydroxylase

Neidigh

Darwanto

Williams

et al. 2009

Chem. Res. Toxicol.

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“…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%

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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“…The 2Ј-hydroxylases have been purified from N. crassa, 156 R. glutanis 149 and A. nidulans 150 and all are able to convert nucleosides with oxygen atoms at C-2 and C-4 of bases. 156, 157 The enzyme from R. glutanis co-purifies with the 7-hydroxylase activity, but genetic evidence suggests that they are separate enzymes. 149…”

Section: Degradation Of 24-dichlorophenoxyacetic Acidmentioning

confidence: 99%