Cloning, expression, and biological function of a dTDP-deoxyglucose epimerase (gerF) gene from Streptomyces sp. GERI-155.
GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules which has antimicrobial activities against gram-positive bacteria. The deoxyhexose biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-155 genome has now been isolated. Four orf were identified and a putative orf, supposed to code for the dTDP-deoxyglucose epimerase gene, was designated as gerF. gerF was expressed in E. coli using recombinant expression vector pHJ3. The recombinant protein expressed in a soluble form. The enzyme was purified by Ni-affinity column using imidazole buffer as eluents. The molecular mass of the expressed protein correlated with the predicted mass (36,000 Da) deduced from the cloned gene sequence data. The purified enzyme produced maltol from dTDP-4-keto-6-deoxyglucose and it was confirmed that the expressed protein was dTDP-deoxyglucose epimerase catalyzing epimerization of C-3 and C-5 or C-3 of dTDP-4-keto-6-deoxyglucose.
Sohng
Jae-Kyung
JK
Institute of Biomolecule Reconstruction, Sun Moon University, Asan, Chungnam 336-708, Korea.
Kim
Hyung-jun
HJ
Nam
Doo-Hyun
DH
Lim
Dong-Ok
DO
Han
Ji-Man
JM
Lee
Hyo-Jung
HJ
Yoo
Jin-Cheol
JC
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry; D002238:Carbohydrate Epimerases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018014:Gene Transfer Techniques; D005947:Glucose / Q000031:analogs & derivatives* / Q000737:chemistry*; D018942:Macrolides / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013942:Thymine Nucleotides / Q000737:chemistry*
D000900:Anti-Bacterial Agents; C102443:GERI 155; D018942:Macrolides; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C112447:dTDP-6-deoxy-4-ketoglucose; D002238:Carbohydrate Epimerases; C487875:dTDP-deoxyglucose epimerase, Streptomyces; D005947:Glucose