Cloning and expression of the glucose-1-phosphate thymidylyltransferase gene (gerD) from Streptomyces sp. GERI-155.
GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules, that has antimicrobial activity against Gram-positive bacteria. The deoxysugar biosynthetic gene cluster of GERI-155 was cloned from Streptomyces sp., GERI-155. One of the orfs, gerD, appeared to encode glucose-1-phosphate thymidylyltransferase (dTDP-glucose synthase), which converts dTTP and glucose-1-phosphate to dTDP-D-glucose and pyrophosphate. GerD was expressed in E. coli in vector pHJ2 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and DEAE-Sepharose CL-6B and DEAE-Trisacryl column chromatography. The specific activity of the enzyme increased 16-fold with a recovery of 10%. It migrated as a single band on SDS-PAGE with a molecular mass of 30 kDa. The purified protein had glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction the highest activity was obtained with the combination of dTTP and alpha-D-glucose-1-phosphate, and only 5.5% of that activity was obtained with UTP in place of dTTP. In the opposite direction the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.
Lee
Hei-Chan
HC
Department of Pharmacy, Chosun University, Kwangju 501-759, Korea.
Sohng
Jae-Kyung
JK
Kim
Hyung-Jun
HJ
Nam
Doo-Hyun
DH
Han
Ji-Man
JM
Cho
Seung-Sik
SS
Choi
Jin-Ho
JH
Yoo
Jin-Cheol
JC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D005810:Multigene Family; D009711:Nucleotides / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; C102443:GERI 155; D018942:Macrolides; D009711:Nucleotides; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase