title: Characterization of LipN (Rv2970c) of Mycobacterium Tuberculosis H37Rv and its Probable Role in Xenobiotic Degradation.
abstract: LipN (Rv2970c) belongs to the Lip family of M. tuberculosis H37Rv and is homologous to the human Hormone Sensitive Lipase. The enzyme demonstrated preference for short carbon chain substrates with optimal activity at 45°C/pH 8.0 and stability between pH 6.0-9.0. The specific activity of the enzyme was 217 U/mg protein with pNP-butyrate as substrate. It hydrolyzed tributyrin to di- and monobutyrin. The active-site residues of the enzyme were confirmed to be Ser216, Asp316, and His346. Tetrahydrolipstatin, RHC-80267 and N-bromosuccinimide inhibited LipN enzyme activity completely. Interestingly, Trp145, a non active-site residue, demonstrated functional role to retain enzyme activity. The enzyme was localized in cytosolic fraction of M. tuberculosis H37Rv. The enzyme was able to synthesize ester of butyric acid, methyl butyrate, in presence of methanol. LipN was able to hydrolyze 4-hydroxyphenylacetate to hydroquinone. The gene was not expressed in in-vitro growth conditions while the expression of rv2970c gene was observed post 6h of macrophage infection by M. tuberculosis H37Ra. Under individual in-vitro stress conditions, the gene was expressed during acidic stress condition only. These findings suggested that LipN is a cytosolic, acid inducible carboxylesterase with no positional specificity in demonstrating activity with short carbon chain substrates. It requires Trp145, a non active site residue, for it's enzyme activity.
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, India.
authors.forename: Dipendrasinh
authors.initials: D
authors.lastname: Jadeja
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, India.
authors.forename: Nandita
authors.initials: N
authors.lastname: Dogra
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, India.
authors.forename: Stuti
authors.initials: S
authors.lastname: Arya
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: Gurdyal
authors.initials: G
authors.lastname: Singh
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, India.
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Kaur
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002087:Butyrates / Q000138:chemical synthesis; D002460:Cell Line; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D004951:Esterification; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006873:Hydroquinones / Q000737:chemistry; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000382:microbiology; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D015262:Xenobiotics / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002087:Butyrates; D004800:Enzymes, Immobilized; D006873:Hydroquinones; D015262:Xenobiotics; C043811:methyl butyrate; D008049:Lipase; C031927:hydroquinone
keywords: 4-HYDROXY PHENYLACETATE HYDROLASE; ESTERASE; LipN; XENOBIOTIC; mycobacterium tuberculosis; rv2970c