Tetrahydrolipstatin Inhibition, Functional Analyses, and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability

Abstract

The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an ␣/␤ hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.

Targeting essential cell wall lipase Rv3802c for potential therapeutics against tuberculosis

“…Sequence analysis M. tuberculosis Rv3802c comprises of 336 amino acids with a molecular mass of 35,448 Da, a lipase with significant phospholipase A and thioesterase activity [19]. Based on the experimental studies on Rv3802c essentiality and its role in the organism's cellular integrity from M. smegmatis, Rv3802c is considered to be good drug target for treating tuberculosis [28,29]. Sequence analysis reveals that Rv3802c is conserved within most of the mycobacterial species including M. leprae and no sequence homolog was found in human (Table S1).…”

“…All these evidences point to the essentiality of Rv3802c. The human lipase inhibitor, tetrahydrolipstatin (http://www.drugbank.ca/drugs/DB01083), inhibits both MSMEG 6394 and Rv3802c with varied degree of inhibition drives us to search for potential inhibitors effective towards mycobacterial proteins [19,28,29].…”

“…Rv3802c is also conserved in Mycobacterium leprae, which is considered a minimal genome [27]. Conditional disruptions of Rv3802c counterpart in Mycobacterium smegmatis leads to the loss of cell wall integrity and internal structure of the cell [28]. All these evidences point to the essentiality of Rv3802c.…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…Interestingly, the Cut4 homologue from Mycobacterium smegmatis is also active against SM (109). M. tuberculosis Cut6, which is cell wall associated (110), also has thioesterase activity and plays a critical role in mycolic acid synthesis, which likely explains why Cut6 is conserved among mycobacteria (113,114). Interestingly, Cut6 induces an immune response in mice which protects them from aerosol challenge with a virulent M. tuberculosis strain (115).…”

Rv3802c in Tuberculosis Therapeutics

“…Conditional disruptions of MSMEG_6394, functional homolog of Rv3802c in M. smegmatis lead to loss of cell wall integrity and internal structure of cell. Recent research effort revealed that tetrahydrolipstatin, a human lipase inhibitor binds irreversibly to both MSMEG_6394 and Rv3802c and exhibting antitubercular activity [10,11]. All these evidences point to the suitability of Rv3802c as promising drug target.…”