Iso-orotate decarboxylase : screening for the gene from Rhodotorula glutinis cDNA library and properties of a synthetic gene

Abstract

Iso-orotate decarboxylase (IDCase) is the final enzyme in the thymine salvage pathway, which is possessed by few organisms. The reaction catalyzed by IDCase is unusual among enzymatic decarboxylases. The IDCase gene from Neurospora crassa has been isolated and transferred to E. coli in the past. But the protein from the E. coli was not produced in large amounts. The IDCase sequence from a Rhodotorula glultinis cDNA library was sought, using gene sequence information from N. crassa compared with the gene of Aspergillus nidulans, to determine the sequence of amino acids that might also be in the IDCase gene sequence of R. glutinis. An IDCase amino acid sequence of 12 amino acids was found to match in the sequence from N. crassa compared with A. nidulans and was used to design an IDCase gene probe. Screening was done for the IDCase geneusing hybridization technique with the R. glutinis cDNA library. However, the sequencing data was not promising. The project shifted to the use of the IDCase gene from Magneportha grisea. Protein isolated from BL21 codon plus cells containing pCal-M. grisea IDCase plasmid was assayed by decarboxylation of the labelled [caroxy-14C] iso-orotate to 14CO2. The assays did not show high activity of the IDCase protein. There is likely a codon bias preventing over-production of the heterologous protein in E. coli, which led to design of a Synthetic IDCase M. grisea gene. On successful cloning, isolation of the protein from BL21 codon plus cells containing the optimized Synthetic pcal-IDCase M. grisea plasmid was done, which showed high activity with [carboxy-14C] iso-orotate radioactivity asssays. The SDS-PAGE gels showed over-expressed IDCase bands at 42.9 kDa. Partial purification of the IDCase protein was done by DEAE Sephacel column chromatography with the protein coming off of the column with salt gradient of 20 mM NaCL to 200 mM NaCl with MOPS lysis buffer, pH6.5. Purification of the IDCase protein would help to carry on further research on the catalytic mechanism of the IDCase enzyme and the cause of rice blast disease by M. grisea.