Iso-orotate decarboxylase : progress toward protein isolation, gene screening, and the non-enzymatic decarboxylation mechanism /

Abstract

Iso-orotate decarboxylase (lDCase) is the final enzyme in the thymine salvage pathway, which is possessed by few organisms. A possible mechanism for this enzymatic decarboxylation is similar to a ~-keto acid decarboxylation, but multiple mechanism schemes are possible for the non-enzymatic decarboxylation reaction. Protein taken from Rhodotorula glutinis cells was assayed for IDCase by decarboxylation of the labeled [carboxy_14C] iso-orotate to 14C02. Attachment of a known inhibitor to an agarose column allowed for a five-fold purification ofIDCase, with a seven percent recovery. A eDNA library of R. glutinis was constructed and converted to a plasmid library to screen for the gene responsible for the IDCase protein. Isolation of the gene responsible for IDCase could help increase effectiveness of IDCase isolation. However, colonies of E. coli grown on restrictive media, designed to select for the presence of IDCase, did not contain active IDCase, according to assay data. For the non-enzymatic decarboxylation, spontaneous decarboxylation of iso-orotic acid does not occur at a measurable rate, but the BU4Am+-iso-orotate salt is decarboxylated at a rate of2.45xlO-6 sec-I. This rate is much slower than the non-published data of iso-orotic acid in water.