Markedly different ascorbate dependencies of the sequential alpha-ketoglutarate dioxygenase reactions catalyzed by an essentially homogeneous thymine 7-hydroxylase from Rhodotorula glutinis.
The alpha-ketoglutarate dioxygenase, thymine 7-hydroxylase (EC 1.14.11.6), has been purified from cultures of Rhodotorula glutinis grown with thymine as a nitrogen source. The purification scheme developed yielded essentially homogeneous preparations of the 7-hydroxylase and also purified another alpha-ketoglutarate dioxygenase, pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3). The purity of the 7-hydroxylase was determined with analytical disc gel electrophoresis in which runs were varied with respect to pH, extent of cross-linking, and the presence of sodium dodecyl sulfate-mercaptoethanol. The 7-hydroxylase apparently exists as a monomer since its molecular weight was 42,700 when determined by molecular gel filtration chromatography and was 40,300 when determined by analytical disc gel electrophoresis under denaturing conditions. Gel filtration chromatography under nondenaturing conditions was used to show that the 2'-hydroxylase has a molecular weight of 64,600. The essentially homogeneous preparations of the 7-hydroxylase were shown to catalyze the thymine-, 5-hydroxymethyluracil-, and 5-formyluracil-dependent oxygenations that are coupled to the decarboxylation of alpha-ketoglutarate, as well as a putative uncoupled decarboxylation which is dependent on uracil. Furthermore, these enzyme preparations were used to show that ATP stimulated the 7-hydroxylase reaction in the absence of ascorbate. Even though it is attractive to consider the four pyrimidine-dependent reactions as being catalyzed by the same active site, they were shown to differ markedly in their dependencies on ascorbate or ATP. The effects of ascorbate and ATP on these reactions, and on the 2'-hydroxylase reaction, are discussed in terms of the possible roles of ascorbate and ATP.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism*; D004589:Electrophoresis, Disc; D003904:Mitosporic Fungi / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008970:Molecular Weight; D010432:Pentoxyl / Q000031:analogs & derivatives / Q000378:metabolism; D012248:Rhodotorula / Q000201:enzymology*; D014498:Uracil / Q000031:analogs & derivatives / Q000378:metabolism
C007777:5-formyluracil; C008055:5-hydroxymethyluracil; D014498:Uracil; D010432:Pentoxyl; D000255:Adenosine Triphosphate; D006899:Mixed Function Oxygenases; C023164:thymine, 2-oxoglutarate dioxygenase; D001205:Ascorbic Acid