Ketohexokinase (KHK), in an adenosine triphosphate (ATP) dependent reaction, catalyzes the first reaction in fructose metabolism, which converts the furanose form of D-fructose into fructose-1-phosphate. This enzyme has become a target for pharmacological development against fatty liver and metabolic syndrome. KHK exists in two isoforms, A and C, which differs by alternative splicing of exon 3 which encodes 45 out of 298 amino acids. Normally KHK exists as a homodimer and is comprised of an alpha/beta domain interlocking with a β-clasp domain. For KHK-C, there appear to be at least two conformations of the β-clasp domain. Previous work on KHK-A reveals it does not adopt the same conformations. A structure of the mouse KHK-A in its unliganded form is solved and shows that these two conformations also exist for KHK-A. Furthermore, this property is conserved across species. While crystals of human KHK-A in its unliganded form were grown, a structure was not achieved. However, unpublished structures of human KHK-A in its unliganded form also shows different conformations in β-clasp domain when in juxtaposition with the same enzyme complex with ligands. Defining the role of conformational changes in KHK-A is important, because this isozyme has been reported to have a role in cancer metastasis.

The thesis describes over-expression, purification and crystallization of three proteins from Staphylococcus aureus (S. aureus). S. aureus is an important human pathogen and methicillin-resistant S. aureus (MRSA) is a serious problem in hospitals nowadays. The crystal structure of 3-Methyladenine DNA glycosylase I (TAG) was determined by single-wavelength anomalous diffraction (SAD) method. TAG is responsible for DNA repair and is an essential gene for both MRSA and methicilin-susceptible S. aureus (MSSA). The structure was also determined in complex with 3-methyladenine (3-MeA) and was solved using molecular replacement (MR) method. An assay was carried out and the molecular basis of discrimination between 3-MeA and adenosine was determined. The native crystal structure of fructose 1-phosphate kinase (PFK) from S. aureus was determined to 2.30 Å and solved using molecular replacement method. PFK is an essential enzyme involved in the central metabolism of MRSA. Despite extensive efforts no co-complex was determined, although crystals were obtained they diffracted poorly. An assay which can be used to test for inhibitors has been developed. Mevalonate Kinase (MK) is another essential enzyme in MRSA and is a key drug target in the mevalonate pathway. Native data diffracting to 2.2 Å was collected. The structure was solved using multiple isomorphorus replacement (MIR) method. A citrate molecule was bound at the MK active site, arising from the crystallization condition. The citrate molecule indicates how substrate might bind. The protein was kinetically characterized. A thermodynamic analysis using fluorescence-based method was carried out on each protein to investigate binding interactions of potential fragments and thus a drug design starting point.