Rv2485c, a Putative Lipase of M. tuberculosis: Expression, Purification and Biochemical Characterization

Abstract

Lipases have been demonstrated to have a role in virulence in several pathogens. Rv2485c gene product of Mycobacterium tuberculosis has been annotated as putative carboxyl esterase (LipQ) involved in cellular metabolism and respiration. The gene was expressed only in oxidative stress condition in in vitro culture of M. tuberculosis H37Ra as shown by Real Time PCR which suggests its role during dormant stage. Thereby, Rv2485c gene was cloned and expressed in E. coli. The LipQ enzyme was purified as a His-tagged protein from inclusion bodies and refolded with 37% protein yield. The specific activity of purified enzyme was calculated to be 93 U/mg with pNP-palmitate as a preferred substrate. It showed optimum enzyme activity in the range of 40-500C and pH 8.0. The Ser-249, Asp-344 and His-377, predicted as the member of the catalytic triad, were confirmed by site directed mutagenesis. The enzyme was inhibited in the presence of PMSF and DEPC suggesting the presence of Ser and His residues in catalytic site. The apparent Km and Vmax were calculated to be 1.45 mM & 196.08 U/ml respectively. The turnover number (kcat) of the enzyme was calculated to be 6.597 min−1. Based on the results it might be suggested that the LipQ is a lipase, hydrolyzing long chain esters, while the expression of gene only in oxidative stress condition suggested that the enzyme might be playing a role in intracellular survival of microorganism in the human macrophages. The manuscript deals with the detail characterization of oxidative stress inducible lipase and represents a step towards the elucidation of its biological function in vivo.