Radioactivity-Based and Spectrophotometric Assays for Isoorotate Decarboxylase: Identification of the Thymidine Salvage Pathway in Lower Eukaryotes

Abstract

A few organisms, notably some fungi, have the ability to metabolize thymidine to uracil, thus conserving the pyrimidine ring for subsequent metabolic use.Neurospora crassapossesses this pathway, termed the thymidine salvage pathway, and can utilize thymidine as a total pyrimidine source. The enzyme isoorotate decarboxylase (IDCase) completes this pathway via the enzymatic removal of the carboxylate from isoorotate to yield uracil. We describe in this communication two assays for IDCase and their application to determine activity levels, kinetic constants, and inhibitory properties. One uses [carboxy-14C]isoorotate from which the enzymatically generated14CO2is collected and quantitated. The second assay utilizes the spectral difference between 2-thioisoorotate and its decarboxylated product, 2-thiouracil. The spectral difference is greatest at 334 nm, out of the range of absorbance of total protein and thus usable for a spectrophotometric assay. The assays are sufficiently sensitive and accurate to be used in the measurement ofKmvalues for both substrates. IDCase activity is found to be significantly higher inN. crassastrains lackinguc-1,a putative regulatory gene, suggesting a degree of metabolic control over this pathway. 5-Nitrouracil is found to inhibit IDCase with an estimatedKivalue that is too low for accurate determination.