Li Chunyan, Li Qiming, Zhang Yuan, Gong Zhen, Ren Sai, Li Ping, Xie Jianping
State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, China.
State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, China.
Microbiol Res. 2017 Mar;196:7-16. doi: 10.1016/j.micres.2016.12.005. Epub 2016 Dec 15.
Lipids and lipases/esterases are essential for Mycobacterium tuberculosis (Mtb) survival and persistence, even virulence. Mycobacterium tuberculosis H37Rv Rv1076 (LipU), a member of lipase family, is homologous to the human Hormone Sensitive Lipase (HSL) based on the presence of conserved motif 'GXSXG'. To define the enzymatic characteristics of rv1076, the gene was cloned, and expressed in Escherichia coli. The protein was purified for enzymatic characterization. LipU showed high specific activity for the hydrolysis of short carbon chain substrates with optimal activity at 40°C/pH 8.0 and stability at low temperature and near-neutral pH. The specific activity, Km and Vmax of LipU was calculated to 176.7U/mg, 1.73μM and 62.24μM/min respectively. Ionic detergents can inhibit its activity. The active-site residues of LipU were determined to be Ser140, Asp244 and His269 by site-directed mutagenesis. The upregulation of Mycobacterium tuberculosis rv1076 under nutritive stress implicates a role in starvation.
脂质和脂肪酶/酯酶对于结核分枝杆菌（Mtb）的存活、持续存在乃至毒力都至关重要。结核分枝杆菌H37Rv的Rv1076（LipU）是脂肪酶家族的一员，基于保守基序“GXSXG”的存在，它与人激素敏感脂肪酶（HSL）同源。为了确定rv1076的酶学特性，该基因被克隆并在大肠杆菌中表达。对该蛋白进行纯化以进行酶学特性分析。LipU对短碳链底物的水解表现出高比活性，在40°C/pH 8.0时具有最佳活性，在低温和近中性pH条件下具有稳定性。LipU的比活性、Km和Vmax分别计算为176.7U/mg、1.73μM和62.24μM/min。离子去污剂可抑制其活性。通过定点诱变确定LipU的活性位点残基为Ser140、Asp244和His269。结核分枝杆菌rv1076在营养应激下的上调暗示其在饥饿中发挥作用。
