RESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Lipase/metabolismo , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Butiratos/síntese química , Linhagem Celular , Estabilidade Enzimática , Enzimas Imobilizadas/química , Esterificação , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Hidroquinonas/química , Cinética , Lipase/química , Lipase/genética , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Especificidade por Substrato , Xenobióticos/química , Xenobióticos/metabolismoRESUMO
rv1922 and rv1923 (lipD) are members of Rv1922-LipD operon in the genome of Mycobacterium tuberculosis. rv1922 was expressed under aerobic and stress conditions, whereas rv1923 was not expressed in aerobically grown bacteria but expressed moderately under oxidative stress conditions. Different expression of both the operonic genes under normal and stress conditions posed apprehensions for the inclusion of rv1922 and rv1923 in the same operon. The results from this study indicated that although the genes were expressed in an operonic manner, there existed the possibility of differential regulation for rv1923 which has been supported by in silico analysis for the presence of putative internal regulatory sites in the operon.