CHARACTERIZATION OF MYCOBACTERIAL ESTRASES/LIPASES USING COMBINED BIOCHEMICAL AND COMPUTATIONAL ENZYMOLOGY

Abstract

Esterases/Lipases are evolutionarily related enzymes mostly belonging to hydrolases superfamily sharing a common a/ß-hydrolase protein fold. Recent studies have suggested that they play a pivotal role in disease manifestation due to disruption of lipid metabolizing enzymes and their pathways, yet only very few have been functionally annotated.

This study focuses on Mycobacterial lipolytic/non-lipolytic enzymes comprising of 31 putative lipases and/or esterases belonging to a/ß-hydrolase fold family. In silico molecular modeling, sequence analysis and ligand docking experiments provide insights into molecular structure of these classes of enzymes. We performed a unique structure based virtual ligand screening to predict natural substrates of 4 putative Mycobacterial esterases/lipases namely BCG_1460c (lipH probable lipase), BCG_2991c (lipN probable lipase/esterase), BCG_2950 (tesA probable thioesterase) and BCG_3229 (lipV possible lipase).

The information obtained from molecular docking and molecular dynamics (MD) simulations was preceded with an in vitro study, where we performed enzymatic assays with whole cell lysates of Mycobacterium bovis BCG over-expressing lipases and/or esterases using synthetic substrates. We provide evidence at structural and biochemical level that the short-chain esterase activity of BCG_1460c (lipH probable lipase) is inhibited by tetrahydrolipstatin (THL) an FDA approved drug. Overall, the in silico and in vitro analysis show high correlation and provide an effective approach to characterize and distinguish mycobacterial lipolytic/non-lipolytic enzymes.