Structural basis of cysteine biosynthesis and peptidoglycan remodelling in Mycobacterium tuberculosis

Mycobacterium tuberculosis is the causative agent of tuberculosis, which is responsible for 1.3 million deaths annually. Treatment is difficult due to an elaborate defense machinery, which includes specific metabolic changes of the bacilli upon exposure to antibiotics and the immune response of the host. The emergence of multi-drug-resistant and extremely drug-resistant strains further complicates the treatment. Therefore the development of novel antibiotics and the investigation of new unexploited targets are of high importance. In the scope of this work, potential drug targets from the L-cysteine biosynthesis and the mycobacterial cell envelope maintenance were investigated. Cysteine biosynthesis provides promising drug targets due to a direct connection between L-cysteine availability and redox homeostasis of the bacilli. Disruption of the mycobacterial redox defense leads to attenuated growth. Three cysteine synthases are encoded in the mycobacterial genome of which CysK1 and CysM have been characterized previously. In this work CysK2 has been enzymatically characterized, catalyzing the formation of L-cysteine from O-phosphoserine and sulfide in a pyridoxal-5-phosphate-dependent reaction. The importance of the cell wall integrity makes it a well exploited target of a broad range of antibiotics. Potent inhibitors and first-line antibiotics are currently in clinical use such as isoniazid and ethambutol, which target biosynthetic pathways of the mycobacterial cell wall. RipA, a protein of the NlpC/p60 family was previously validated as essential for infectivity. The characterized NlpC/p60 proteins are peptidoglycan hydrolases and are involved in cell division and peptidoglycan recycling. In M. tuberculosis, four proteins represent this family: RipA, RipB, RipC and RipD. Here we report the biochemical and biophysical characterization of RipA, RipB and RipD as well as their high resolution structures. The detailed understanding of these enzymes prepares the ground for structure-based inhibitor development ...