Characterization and function of Mycobacterium tuberculosis H37Rv Lipase Rv1076 (LipU).
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.
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.
Chunyan
C
Li
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.
Qiming
Q
Li
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.
Yuan
Y
Zhang
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.
Zhen
Z
Gong
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.
Sai
S
Ren
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.
Ping
P
Li
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. Electronic address: georgex@swu.edu.cn.
Jianping
J
Xie
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D004950:Esterases; D008049:Lipase
Characteristics; Lipases/esterases; Mycobacterium tuberculosis; Rv1076; lipU