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The deoxysugar biosynthetic gene cluster of Sch47554/Sch 47555 was cloned from Streptomyces sp. SCC-2136. One of the ORFs, schS6, appeared to encode glucose-1-phosphate thymidylyltransferase, which converts dTTP andglucose-1-phosphate to TDP-D-glucose and pyrophosphate.The dTDP-D-glucose is a key metabolite in prokaryotics as aprecursor for a large number of modified deoxysugars, andfrom glycosides to macrolides. SchS6 was expressed in E. colivector pSCHS6 and the expressed protein was purified toaparent homogeneity by ammonium sulfate precipitationand Ni-NTA afinity column chromatography. The specificactivity of the purified enzyme increased 4.7-fold with 17.5%recovery. It migrated as a single band on SDS-PAGE with anaparent molecular mas of 56 kDa. The purified proteinshowed glucose-1-phosphate thymidylyltransferase activity,the forward reaction, the highest activity was obtained withcombination of dTTP and α-D-glucose-1-phosphate, andonly 12% of that activity was obtained with the substratesUTP/α-D-glucose-1-phosphate. In the oposite direction, thepurified protein was highly specific for dTDP-D-glucose andpyrophosphate.