Cloning and expression of glucose-1-phosphate thymidylyltransferase gene (schS6) from Streptomyces sp. SCC-2136.
The deoxysugar biosynthetic gene cluster of Sch 47554/Sch 47555 was cloned from Streptomyces sp. SCC-2136. 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, and these deoxysugars are a major part of various antibiotics, ranging from glycosides to macrolides. SchS6 was expressed in E. coli vector pSCHS6 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and Ni-NTA affinity column chromatography. The specific activity of the purified enzyme increased 4.7-fold with 17.5% recovery. It migrated as a single band on SDS-PAGE with an apparent molecular mass of 56 kDa. 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 alpha-D-glucose-1-phosphate, and only 12% of that activity was obtained with the substrates UTP/alpha-D-glucose-1-phosphate. In the opposite direction, the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.
Han
Ji-Man
JM
Department of Biochemistry and Molecular Biology, University of Ulsan, College of Medicine, Seoul 138-736, Korea.
Kim
Su-Min
SM
Lee
Hyo-Jung
HJ
Yoo
Jin-Cheol
JC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics*; D013379:Substrate Specificity
D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase