Lysine-322 Acetylation Negatively Regulates Isocitrate Lyase of <em>Mycobacterium tuberculosis</em>

doi:10.13523/j.cb.20150602

China Biotechnology

2015, Vol. 35

Issue (6): 8-13 DOI: 10.13523/j.cb.20150602

Lysine-322 Acetylation Negatively Regulates Isocitrate Lyase of Mycobacterium tuberculosis

LI Yao-yao, BI Jing, WANG Yi-hong, QIN Yun-he, ZHANG Xue-lian

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China

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Abstract

Objective: Isocitrate lyase (ICL) of Mycobacterium tuberculosis (Mtb) was acetylated at lysine 322. The aim was to investigate ICL Lys322 acetylation and its regulatory function in activity of ICL. Methods: The prokaryotic expression plasmids pET28a-icl was constructed and Lys322 of ICL was site-directed mutated into arginine (Arg, R) and glutamine (Gln, Q), respectively. Then recombinant proteins ICL^WT,ICL^322R and ICL^322Qwere expressed and purified in vitro. Acetylation of wild type ICL and its mutants were examined by Western blotting with anti-acetyllysine antibody, and enzyme activities of proteins were evaluated. Results: Western blotting showed that ICL^WT,ICL^322R and ICL^322Q were indeed acetylated proteins. Compared with activity of ICL^WT, mutation of Lys322 to arginine decreased the enzyme activity by approximately 50%, and glutamine substitution dramatically decreased the activity more than 70%. Conclusion: The results of Western blotting showed that the ICL and two mutant proteins can be acetylated effectively in E. coli expression system. Decreasing in ICL^322Q activity suggested that Lys322 acetylation could negatively regulate isocitrate lyase of Mycobacterium tuberculosis. This provided a foundation to study an important aspect of Mtb, the metabolic regulation of Mtb, which may lead to new knowledge on latent infection.

Key words： Isocitrate lyase acetylation site-directed mutagenesis

Received: 13 March 2015 Published: 25 June 2015

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LI Yao-yao, BI Jing, WANG Yi-hong, QIN Yun-he, ZHANG Xue-lian. Lysine-322 Acetylation Negatively Regulates Isocitrate Lyase of Mycobacterium tuberculosis. China Biotechnology, 2015, 35(6): 8-13.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20150602 OR https://manu60.magtech.com.cn/biotech/Y2015/V35/I6/8

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