Cloning and characterization of CalS7 from Micromonospora echinospora sp. calichensis as a glucose-1-phosphate nucleotidyltransferase.
The deoxysugar biosynthetic gene cluster of calicheamicin contains the calS7, which encodes glucose-1-phosphate nucleotidyltransferase and converts glucose-1-phosphate and nucleotides (NTP) to NDP-glucose and pyrophosphate. calS7 was expressed in Escherichia coli BL21(DE3), and the purified protein had significant thymidylyltransferase and uridylyltransferase activities as well, with some guanidylyltransferase activity but negligible cytidyl and adenyltransferase activity. The functions of thymidylyltransferase and uridylyltransferase were also verified using one-pot enzymatic synthesis of TMK and ACK. The products were analyzed by HPLC and ESI/MS, which showed peaks at m/z = 563 and 565 for TDP-D: -glucose and UDP-D-glucose, respectively, in negative mode.
Simkhada
Dinesh
D
Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, # 100 Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Republic of Korea.
Oh
Tae-Jin
TJ
Kim
Eui Min
EM
Yoo
Jin Cheol
JC
Sohng
Jae Kyung
JK
D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005958:Glucosephosphates / Q000378:metabolism*; D008847:Micromonospora / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization
D000617:Aminoglycosides; D001426:Bacterial Proteins; D005958:Glucosephosphates; D009711:Nucleotides; C031590:glucose-1-phosphate; D009713:Nucleotidyltransferases