Expression and characterization of the carboxyl esterase Rv3487c from Mycobacterium tuberculosis.
Rv3487c (lipF), a member of the lipase family of Mycobacterium tuberculosis, is related to virulence of this pathogen. Real-time RT-PCR analysis indicated that Rv3487c was induced at low pH in M. tuberculosis cultured in vitro. The gene of Rv3487c was cloned and expressed as fusion protein in Escherichia coli. After removal of the N-terminal domain of the fusion partner by enterokinase treatment, the effect of pH, temperature, and detergents on the purified enzyme activity and stability was characterized. Rv3487c could efficiently hydrolyze short chain esters. The catalytic triad of Rv3487c consists of residues Ser90, Glu189, and His219 as demonstrated by amino acid sequence alignment, three-dimensional modeling, and site-directed mutagenesis.
Zhang
Min
M
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, PR China.
Wang
Jia-dong
JD
Li
Zuo-feng
ZF
Xie
Jun
J
Yang
Yan-ping
YP
Zhong
Yang
Y
Wang
Hong-hai
HH
D000595:Amino Acid Sequence; D043182:Carboxylesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004765:Enteropeptidase / Q000737:chemistry; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics; D013696:Temperature; D013879:Thioredoxins / Q000235:genetics
D003902:Detergents; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D012694:Serine; D006639:Histidine; D013879:Thioredoxins; D043182:Carboxylesterase; D004765:Enteropeptidase