ORIGINAL RESEARCH PAPER Cloning and characterization of CalS7 from Micromonospora echinospora sp. calichensis as a glucose-1-phosphate nucleotidyltransferase Dinesh Simkhada Tae-Jin Oh Eui Min Kim Jin Cheol Yoo Jae Kyung Sohng Received: 22 May 2008 / Revised: 28 August 2008 / Accepted: 29 August 2008 / Published online: 20 September 2008 Ó Springer Science+Business Media B.V. 2008 Abstract The deoxysugar biosynthetic gene cluster of calicheamicin contains the calS7, which encodes glucose-1-phosphate nucleotidyltransferase and con- verts 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 gua- nidylyltransferase activity but negligible cytidyl and adenyltransferase activity. The functions of thy- midylyltransferase 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. Keywords Calicheamicin  Deoxysugar  Micromonospora echinospora sp. calichensis  Thymidylyltransferase  Uridylyltransferase Introduction Calicheamicin produced by Micromonospora echinos- pora ssp. calichensis is an endiyne antibiotic containing three distinct structural elements: a DNA-recognition unit, which delivers the metabolite to its target DNA; an activation component, which sets the stage for cyclo- aromatization; and the enediyne ‘‘warhead’’, which cycloaromatizes to a highly reactive diradical species in the presence of DNA (Myers et al. 1994; Biggins et al. 2003) (Fig. 1). The basic property of calicheamicin resides is its ability to cleave DNA. This cleavage property can be triggered by the action of a thiol- reducing agent or UV exposure. The enediyne moiety plays an important role in DNA cleavage, while the sugar parts are supposed to help the antibiotic become more specific or direct it to bind the double-stranded DNA. In this sense, the all-structural constituents are equally important (Biggins et al. 2003; Galm et al. 2005). The nucleotide-sugar NDP-D-glucose is a key metabolite in prokaryotes where it serves as a precursor to a large number of modified sugars, such as L-rhamnose (6-deoxyhexose), 6-deoxy-L-talose, 2,6-dideoxyhexose and other deoxyhexoses and deoxypentose (Simone et al. 2001). Numerous stud- ies have demonstrated that the early enzymatic step common to the biosynthesis of all deoxysugars found in antibiotics is the formation of NDP-glucose from NTP and a-D-glucose-1-phosphate from glucose- 1-phosphate nucleotidylyltransferase (NDP-glucose D. Simkhada  T.-J. Oh  E. M. Kim  J. K. Sohng (&) Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, # 100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Republic of Korea e-mail: sohng@sunmoon.ac.kr J. C. Yoo Department of Pharmacy, College of Pharmacy, Chosun University, Gwangju 501-759, Korea 123 Biotechnol Lett (2009) 31:147–153 DOI 10.1007/s10529-008-9844-9