Structural Studies Targeting Enzymes Involved in the Biosynthesis of Complex Cell Surface Lipids of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) is a deadly pathogen that causes the infectious diseases Tuberculosis (TB) in humans. The emergence of drug resistant TB has created an urgent need for the development of new chemotherapeutics that can counter the resistance, and combat the spread of TB by shortening the treatment. One of the major determinants of the virulence of Mtb is its thick cell envelope which contains a wide variety of complex lipids and carbohydrates. Thus, the enzymes of cell wall lipid biosynthesis in Mtb represent attractive targets for drug discovery. In this dissertation, our studies on the structural and functional characterization of three different proteins from complex lipid synthesis pathways in the Mtb are presented.

Firstly, we have crystallized the 225 kDa methyl-branched fatty acid synthesizing mycocerosic acid synthase (MAS) from Mtb. We developed methods to improve the diffraction properties of the MAS crystals. A medium resolution dataset allowed initial crystallographic characterization of MAS. The results suggest that these methods could be adapted for crystallographic studies of other type I polyketide synthase from Mtb.

Secondly, the crystal structure of Mtb acyl carrier protein synthase (AcpS) is presented. This important enzyme activates the acyl carrier protein domains of the FAS I and FAS II systems in Mtb. The structure revealed that the Mtb AcpS adopts two pH dependent different conformations – open and closed. In vitro studies established a structure-function relationship by demonstrating a pH dependent activity profile for AcpS.

Thirdly, we have determined the structural basis for the inhibition of the thioesterase (TE) domain of Pks13 by benzofuran class of inhibitors. Pks13 represents a novel target in the essential mycolic acid biosynthesis pathway in Mtb. The crystal structure established that the TE domain belonged to the α/β-hydrolase class of enzymes. We also determined the structural basis for the mechanism of resistance of TE domain towards these inhibitors. We used the structure-guided approach to design new analogs of the benzofuran scaffold, and our best analog exhibited 12-fold increased inhibitory activity against Mtb. We also established that the benzimidazole scaffold containing analogs bind to the TE domain in a manner similar to the benzofurans.