The respiratory disease tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (Mtb), is an ongoing worldwide epidemic that necessitates the identification of novel drug targets. Sulfur is an essential element for the growth, virulence, and survival of Mtb, and thus disruption of sulfur metabolism may be a potential means of combating TB. This dissertation discusses research conducted on CysDNC, the key sulfate activating complex (SAC) that lies at the start of the mycobacterial sulfur pathway. Sulfur metabolism begins when intracellular sulfate is reacted with adenosine 5′-triphosphate (ATP) to form the product adenosine 5’-phosphosulfate (APS). This reaction is catalyzed by the ATP sulfurylase CysD and is energetically coupled to hydrolysis of GTP by the GTPase domain of CysNC. APS is then further phosphorylated into 3’-phosphoadenosine-5’-phosphosulfate (PAPS) by the APS kinase domain of CysNC. The products APS and PAPS serve as precursors for downstream sulfur-containing biomolecules. The individual subunits CysD and CysNC associate together to form the trifunctional CysDNC complex. In this dissertation, CysDNC has been characterized by several approaches.
The opening chapter of this work reviews the role of sulfur-containing compounds in TB infection, previous work done on bacterial SACs, and sulfate-activating enzymes from various species across the kingdom of life. Chapter 2 proceeds with initial observations on the structure and stability of CysDNC during recombinant purification of the complex from the expression host Escherichia coli as well as M. smegmatis, a species closely related to M. tuberculosis. Kinetic studies of CysDNC are discussed in Chapter 3. The activity of CysDNC in the reverse direction of the ATP sulfurylase reaction was characterized in the absence and presence of GTP and GDP. Two other kinetic assays, a commercially-available pyrophosphate detection assay and a “molybdolysis” assay that aimed to decouple the ATP sulfurylase and GTPase activities, were also tested. Chapter 4 covers structural characterization of the CysDNC complex using several methods, including X-ray crystallography, cryogenic electron microscopy, and SEC-SAXS-MALS. Results of experiments conducted with these three methods all point towards a higher-order CysDNC oligomer that exhibits a large degree of flexibility and conformational variability. Chapter 5 examines the growth phenotype of an M. smegmatis mutant strain deficient in CysD (ATP sulfurylase). This mutant was constructed using a mycobacterium-specific recombination-mediated genetic engineering method. The mutant cannot grow in liquid culture when sulfate is present as the sole sulfur source, but it is able to utilize reduced sulfur compounds for growth. Further, the mutant shows increased sensitivity to common anti-TB drugs.
The two remaining chapters in this dissertation discuss two shorter collaborative projects united by the common theme of structural biology. Chapter 6 presents the crystal structure of iSeroSnFR, an engineered genetically-encoded fluorescent serotonin sensor. The structure of the sensor in its ligand-free, open conformation is described and compared with those of other similar sensors. Attempts to co-crystallize iSeroSnFR with its ligand serotonin are also discussed. Chapter 7 discusses X-ray crystallographic work on FtsZ, a cytokinetic bacterial GTPase. FtsZ is an important target for the development of new antibiotic drugs, as it forms the Z-ring, a constricting ring-like structure that is critical for the separation of daughter cells. To facilitate fragment-based drug discovery via covalent tethering, crystallography was conducted for cysteine mutants of FtsZ from the bacteria Bacillus subtilis and Pseudomona aeruginosa. Also discussed are attempts to co-crystallize P. aeruginosa wildtype FtsZ with potential inhibitors identified from a computational drug screen. Preliminary kinetic work presented in this chapter shows that some of the computational hits do indeed inhibit FtsZ.
