Preliminary characterization of IDCase in Neurospora crassa and other fungi organisms.

Abstract

Preliminary characterization of isoorotate decarboxylase (IDCase) in Neurospora crassa and other fungi organisms was determined by a radioactivity-based assay using the radioactive isotope carbon 14 to label the substrate (isoorotate, IOA) at C-7. A crude enzyme preparation was obtained from Neurospora crassa which stoichiometrically converts IOA to uracil and carbon dioxide (C02), The decarboxylation assay measures the amount of IDCase activity within a protein preparation by detecting the amount of 14CO2 released from IDA in a single reaction at a given time point. Wild-type (74A) N. crassa had a specific enzyme activity of 0.10 nmol/min/mg which was not significantly different when the organism was grown in the presence of uracil. Mutant strains of N. crassa that lack certain thymidine salvage enzymes were obtained and tested to observe if whether IDCase levels can be altered with changes of pyrimidine and nitrogen sources in the growth medium. The mutant strain 2203 showed a 3-fold increase in specific enzyme activity when it was grown in the presence of uracil and about a I5-fold increase in the presence of thymine. The labeled strain 2204 showed more than a 2-fold increase in the presence of uracil. When compared to wildtype Aspergillus nidulans showed more than a I-fold increase in specific enzyme activity. The other organisms assayed for IDCase activity did not have a significant amount of specific enzyme activity. Chemically similar compounds were tested as inhibitors ofIDCase. 5-HU and 5-FU inhibits about 40% of the enzyme activity. Although 5-NO2U inhibits almost all of the enzyme activity. The results of the assay indicate that this organism and others (where noted) utilize the thymidine salvage pathway to salvage and synthesize pyrimidines needed for ribonucleic acid (RNA), hence deoxyribonucleic acid (DNA) synthesis.