Isolation and characterization of thymine-7-hydroxylase from Rhodotorula glutinis and iso-orotate decarboxylase from Neurospora crassa

Abstract

"N. crassa" and "R. glutinis" are known to utilize a unique salvage pathway for the production of uracil. This is known as the pryimidine salvage pathway. This pathway consists of four enzymatic steps to convert thymidine to uracil. The third enzyme of the pathway is thymine-7-hydroxylase (THase), which forms uracil-5-carboxylic acid from thymine by way of three oxidative reactions. The final enzyme is iso-orotate decarboxylase (IDCase), which forms uracil from iso-orotate through a decarboxylation reaction. The THase gene was transferred into an expression vector. Multiple culture condition were used to optimize protein production. THase activity was measured using radioactive [14C] thymine assays. The iso-orotate decarboxylase (IDCase) gene was also transferred into an expression system. Multiple culture condition were used to optimize protein production. Various protein purification methods were used in an attempt to purify IDCase. The conserved cysteine residue Cys272 was changed by site-directed mutagenesis to Ala272; the mutant protein had only slightly diminished activity.