Overexpression and substrate specificity studies of phosphodeoxyribomutase and thymidine phosphorylase

Abstract

The gene encoding phosphodeoxyribomutase and thymidine phosphorylase [EC 2.4.2.4] from Escherichia coli were excised from plasmid pVH19 utilizing AvaI restriction enzyme and ligated to the expression vector pKK-226 containing the E. coli tac promoter. Introduction of the resulting plasmid pCB-11 into E. coli JM105 allowed for the overexpression of both enzymes upon induction with isopropylthiogalactoside. A 3-L fermentation of this E. coli strain produced 1500 and 75,000 units, respectively, of the mutase and nucleoside phosphorylase. Substrate specificity studies indicate that the mutase accepts d-ribose 5-phosphate and d-arabinose 5-phosphate as substrates in addition to its natural substrate 2-deoxyribose 5-phosphate. Thymidine phosphorylase showed, however, narrow substrate specificity on the pentose moiety. Only α- d-2-deoxyribose 1-phosphate was accepted as substrate. α- d-Ribose 1-phosphate and α- d-arabinose 1-phosphate are not acceptable based on a coupled enzymatic assay. d2,3-Dideoxyribose 5-phosphate was not converted to nucleoside based on the coupled enzymatic reactions using phosphodeoxyribomutase and thymidine or purine nucleoside phosphorylase.