title: Characterization of a novel esterase Rv1497 of Mycobacterium tuberculosisH37Rv demonstrating β-lactamase activity.
abstract: The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothetical esterases and penicillin binding proteins ofM. tuberculosis as well as to be involved in lipid metabolism. Sequence alignment revealed that Rv1497 protein contains characteristic consensus β-lactamase motif 'SXXK' in addition to a conserve pentapeptide -GXSXG-, characteristic of lipolytic enzymes, at the C-terminus of protein in contrast to its usual N-terminus location. For detailed characterization of protein, the rv1497 gene was cloned, expressed with N-terminal His-tag and purified to homogeneity on Ni-NTA column. Rv1497 demonstrated both esterase and β-lactamase activities. A serine located within consensus β-lactamase motif 'SXXK' was identified as catalytic residue in both esterase and β-lactamase enzymatic activities whereas serine residue located within conserved pentapeptide did not show any effect on both enzyme activities. The catalytic residues of Rv1497 for β-lactamase activity were determined to be Ser88, Tyr-175 and His355 residues by site-directed mutagenesis. The enzyme demonstrated preference for short chain esters (pNP-butyrate). The expression of lipL gene was significantly up-regulated during acidic stress as compared to normal conditions in in vitro culture of M. tuberculosis H37Ra. This is perhaps the first report demonstrating an esterase of mycobacterium showing β-lactamase activity.
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, India.
authors.forename: Gurpreet
authors.initials: G
authors.lastname: Singh
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, India.
authors.forename: Arbind
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, India.
authors.forename: Stuti
authors.initials: S
authors.lastname: Arya
authors.affiliation: National JALMA Institute for Leprosy & Other Mycobacterial Diseases, Agra, India.
authors.forename: Umesh Dutt
authors.initials: UD
authors.lastname: Gupta
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, India.
authors.forename: Kashmir
authors.initials: K
authors.lastname: Singh
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, India. Electronic address: jagsekhon@yahoo.com.
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Kaur
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000667:Ampicillin / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004790:Enzyme Induction; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D015854:Up-Regulation; D001618:beta-Lactamases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000667:Ampicillin; D001618:beta-Lactamases
keywords: Esterase; Mycobacterium; Rv1497; β-Lactamase