Overexpression, purification and truncation analysis of RmlC protein of Mycobacterium tuberculosis.
- Author:
Jong Seok LEE
1
;
Tae Yoon LEE
;
Jae Ho PARK
;
Jong Sun KIM
;
Tae Jin LEE
;
Jai Youl LEE
;
Sung Kwang KIM
Author Information
1. Department of Microbiology, College of Medicine, Yeungnam University, 317-1 Daemyung-5-dong, Nam-gu, Taegu, 705-035, South Korea. doxr7p@medical.yeungnam.ac.kr
- MeSH:
Cell Wall;
Mycobacterium tuberculosis*;
Mycobacterium*;
Peptidoglycan;
Tuberculosis
- From:Journal of the Korean Society for Microbiology
2000;35(4):273-282
- CountryRepublic of Korea
- Language:Korean
-
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-1-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 DELTArmlC E. coli strain SPHI874 (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.
