Cloning and Characterization of Rhodotorula glutinis Thymine Hydroxylase

被引:13
作者
Neidigh, Jonathan W. [1 ]
Darwanto, Agus [1 ]
Williams, Adides A. [1 ]
Wall, Nathan R. [1 ]
Sowers, Lawrence C. [1 ]
机构
[1] Loma Linda Univ, Sch Med, Dept Basic Sci, Loma Linda, CA 92350 USA
关键词
KETOGLUTARATE-DEPENDENT DIOXYGENASE; URACIL-DNA GLYCOSYLASE; 5-HYDROXYMETHYLURACIL-DNA GLYCOSYLASE; OXIDATIVE DEMETHYLATION; SUBSTRATE-SPECIFICITY; ASPERGILLUS-NIDULANS; NEUROSPORA-CRASSA; 7-HYDROXYLASE; DAMAGE; PROTEIN;
D O I
10.1021/tx8004482
中图分类号
R914 [药物化学];
学科分类号
100701 ;
摘要
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 arc 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.
引用
收藏
页码:885 / 893
页数:9
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