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 thym...

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Published in:Molecules and cells 2004-04, Vol.17 (2), p.274-280
Main Authors: Lee, Hei-Chan, Sohng, Jae-Kyung, Kim, Hyung-Jun, Nam, Doo-Hyun, Han, Ji-Man, Cho, Seung-Sik, Choi, Jin-Ho, Yoo, Jin-Cheol
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Base Sequence
Cloning, Molecular
Enzyme Inhibitors - metabolism
Macrolides - chemistry
Macrolides - metabolism
Molecular Sequence Data
Molecular Structure
Molecular Weight
Multigene Family
Nucleotides - metabolism
Nucleotidyltransferases - genetics
Nucleotidyltransferases - metabolism
Open Reading Frames
Sequence Alignment
Sequence Homology, Amino Acid
Streptomyces - enzymology
Streptomyces - genetics
Substrate Specificity
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Summary: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.
ISSN:1016-8478
DOI:10.1016/s1016-8478(23)13038-x