Cloning and characterization of CalS7 from Micromonospora echinospora sp. calichensis as a glucose-1-phosphate nucleotidyltransferase
- 205 Downloads
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.
KeywordsCalicheamicin Deoxysugar Micromonospora echinospora sp. calichensis Thymidylyltransferase Uridylyltransferase
This study was supported by a grant of the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (0720260) and Korean Research Foundation (KRF) by providing research scholarship for a Ph D scholar.
- Aragäo D, Marques AR, Frazäo C et al (2006) Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate. Acta Crystallogr Sect F Struct Biol Cryst Commun 62:930–934PubMedCrossRefGoogle Scholar
- Kieser T, Bibb MJ, Buttner MJ et al (2000) Practical streptomyces genetics. John Innes Foundation, NorwichGoogle Scholar