Streptomyces sp. SCC-2136의 deoxysugar 생합성에 관련된 Glucose-1-Phosphate Thymidylyltransferase 유전자(schS6)의 과발현 단백질 분리정제 및 생화학적 특성연구

Abstract

The deoxysugar biosynthetic gene cluster of Sch 47554/ Sch 47555 was cloned from Streptomyces sp. SCC-2136. The three orfs namely schS6, schS5, and schS2 were amplified, cloned and heterologously expressed in E. coli. One of the orfs, schS6, appeared to encode glucose-1-phosphate thymidylyltransferase, which converts dTTP and Glucose-1-Phosphate to TDP-D-glucose and pyrophosphate. The dTDP-D-glucose is a key metabolite in prokaryotics as a precursor for a large number of modified deoxysugars. These deoxysugars are major part of various antibiotics from glycosides to macrolides. SchS6 was expressed in E.coli in the vector pSCHS6 and the expression protein was purified to apparent homogeneity by ammonium sulfate precipitation and Ni-NTA affinity column using imidazole buffer as eluents. The specific activity of the enzyme increased 4.7-fold with a recovery of 17.5%. It migrated as single band on SDS-PAGE with a molecular mass of 56kDa. The purified protein showed glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction the highest activity was obtained with combination of dTTP and α-D-glucose 1-phosphate, and only 12% of that activity was obtained with the substrates UTP/α-D-glucose 1-phosphate. And the purified protein was highly specific for dTDP-D-glucose and pyrophosphate in the reverse reaction.