Structural enzymology of dormancy related proteins from Mycobacterium tuberculosis

Abstract: Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most lethal infectious agents affecting humans. The World Health Organisation estimates that about one-third of the world s population is infected with persistent mycobacteria and that tuberculosis accounts for approximately 2 million deaths annually. Treatment of this disease is complicated by the ability of Mycobacterium tuberculosis to switch from the active form into a persistent phase. In this phase the bacteria have little or no replication and are not targeted as effectively by the antibiotics used today. The emergence of multi-drug resistant and extensively drug resistant strains of Mycobacterium tuberculosis poses a major threat to human health worldwide. Therefore the development of novel antibiotics especially targeting Mycobacterium tuberculosis in the persistent state is of highest interest. In this thesis three target proteins from persistent phase Mycobacterium tuberculosis have been studied by protein crystallography and biochemical methods. The studies have focused on CysM, a cysteine synthase where transposon knockouts are attenuated in macrophages and AlaDH, a secreted protein showing altered expression profile upon adaptation to dormancy. Furthermore NarL, a member of the two component regulatory system family was studied since it has been shown to be up-regulated in dormant bacteria. This work has been done within a large consortium with the aim to develop novel antibiotics which are active against stationary phase persistent Mycobacterium tuberculosis. The three-dimensional structure and subsequent functional characterization of CysM revealed that the enzyme is an O-phosposerine dependent sulfhydrylase in contrast to previous annotations as an O-acetylserine sulfhydrylase. With these findings we can conclude that CysM is part of a new cysteine biosynthesis pathway that is independent of both O-acetylserine and sulphide. Furthermore it was shown that the C-terminus of CysM is acting as a lid and thereby protecting the reaction intermediate from spontaneous nucleophilic attack. The C-terminus is also responsible for the specificity of the thiocarboxylated protein substrate used in the second half reaction. The structures of apo and holo-AlaDH revealed a large domain rotation upon nucleotide binding. In the structure of the abortive complex with NAD+ and pyruvate, the substrates are suitably placed for hydride transfer between the nicotinamide ring and pyruvate. As the abortive complex that we have trapped most likely represents an active conformation of the enzyme it can serve as a suitable template for inhibitor design. The structure of the N-terminal domain of NarL was determined to 1.9Å and is similar to the structures of previously determined response regulators. Additionally a key residue is identified which might play an important role for the stability of the phospho-aspartyl anhydride. In conclusion this thesis describes the structure and functional characterization of three potential drug targets from persistent phase tuberculosis.