Cloning and Characterization of Rhodotorula glutinis Thymine Hydroxylase

Department of Basic Sciences, Loma Linda University School of Medicine, Alumni Hall for Basic Science, Room 101, 11021 Campus Street, Loma Linda, California 92350, USA.
Chemical Research in Toxicology (Impact Factor: 3.53). 05/2009; 22(5). DOI: 10.1021/tx8004482


Thymine hydroxylase (TH) is a member of the α-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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    • "THase ) , also a member of 2OG - dependent dioxygenases , acts as a key enzyme in the thymidine salvage pathway in fungi ( e . g . , Neurospora crassa ) and catalyzes three sequential oxidation reactions to convert thymine ( T ) to iso - orotate , which is subse - quently processed by a decarboxylase to produce uracil ( U ) ( Smiley et al . 2005 ; Neidigh et al . 2009 ) . Based on the similar chemistry between 5mC oxidation and the T - to - U conversion , we previously hypothesized that , in theory , Tet proteins should be able to further oxidize 5hmC to 5 - formylcytosine ( 5fC ) and 5 - carboxylcytosine ( 5caC ) ( Wu and Zhang 2010 ) . However , previous studies failed to detect these predicted enz"
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