The Journal of the Korean Society for Microbiology (대한미생물학회지)

The Korea Society for Microbiology (대한미생물학회)
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Overexpression, Purification and Truncation Analysis of RmlC Protein of Mycobacterium tuberculosis

  • Lee, Jong-Seok (Department of Microbiology, College of Natural Sciences, Kyungpook National University) ;
  • Lee, Tae-Yoon (Department of Microbiology, College of Medicine, Yeungnam University) ;
  • Park, Jae-Ho (Department of Internal Medicine, Keimyung University School of Medicine) ;
  • Kim, Jong-Sun (Department of Microbiology, College of Medicine, Yonsei University) ;
  • Lee, Tae-Jin (Department of Microbiology, College of Natural Sciences, Kyungpook National University) ;
  • Lee, Jai-Youl (Department of Microbiology, College of Natural Sciences, Kyungpook National University) ;
  • Kim, Sung-Kwang (Department of Microbiology, College of Medicine, Yeungnam University)
  • Published : 2000.08.31
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Abstract

dTDP-rhamnose provides L-rhamnose to the bridge-like structure between mycolyl arabinogalactan and peptidoglycan of the mycobacterial cell wall. dTDP-rhamnose is composed of glucose-l-phosphate and dTTP by four enzymes encoded by rmlA-D. To determine the region(s) of RmlC protein essential for its dTDP-4-keto-6-deoxyglucose epimerase activity, we overexpressed both whole (202 amino acids) and three different truncated (N-terminal 106 or 150 or C-terminal 97 amino acids) RmlC proteins of Mycobacterium tuberculosis. The RmlC enzyme activity in the soluble lysates of ${\Delta}rmlC$ E. coli strain $S{\Phi}874$ (DE3 PlysS) expressing the wild type or truncated rmlC genes was initially analyzed by three sequential reactions from dTDP-glucose to dTDP-rhamnose in the presence of purified RmlB and RmlD. All three soluble lysates containing the truncated RmlC proteins showed no enzyme activity, while that containing the wild type RmlC was active. This wild type RmlC was then overexpressed and purified. The incubation of the purified RmlC enzyme so obtained with dTDP-4-keto-6-deoxyglucose resulted in the conversion of dTDP-4-keto-rhamnose. The results show that the truncated regions of the RmlC protein are important for the RmlC enzyme activity in M. tuberculosis.

Keywords

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