Part I. Recently there has been an alarming rise in the number of infections due to pathogenic organisms that are insensitive to our current arsenal of pharmaceuticals, necessitating the identification of new antimicrobial targets. Here, we describe structural studies of two enzymes from the aspartate family of biosynthetic enzymes in order to assess their potential for drug targeting. Our first report details how an unlikely inhibitor with low millimolar binding characteristics, 5-hydroxy-4-oxo-norvaline, can effectively inactivate homoserine dehydrogenase HSD) through the formation of a tight-binding covalent adduct. This is followed by the structural characterization of the first phenolic-based non-amino acid inhibitor of HSD, which is shown to occupy the substrate binding site and make specific contacts with residues involved in catalysis. Together, these studies lay the foundation for further structure-based drug design of novel inhibitors of HSD. Following this, we report the first structure of the next enzyme in the aspartate pathway, homoserine transacetylase HTA). As the committal step in microbial methionine biosynthesis, this enzyme is essential to microbial survival. The high-resolution crystal structure identifies HTA as a new member of a larger structural family of enzymes known as the alpha/beta hydrolases. Using the structure as a guide, we propose a rationale for the previously reported ping-pong reaction mechanism for this enzyme. We conclude with a preliminary look into how the natural substrate, homoserine, binds in the active site and some subtle differences between HTAs from different sources. Part II. Microbes have long been admired for their ability to process virtually any chemical. In Part II of this work we will engage in the structural characterization of two enzymes involved in the microbial degradation of xenobiotic compounds. The first enzyme, cyclohexylamine oxidase CHAO), is the enabling step in the bacterial metabolism of cyclohexylamine. Preliminary crystallographic analysis of cofactor bound and ternary complexes of CHAO, will be used to highlight differences between this enzyme and its closest human homologue. This information will then be used to direct structure inspired mutagenesis studies in order to better understand the substrate specificity of this enzyme and how it might be altered. Finally, the last enzyme to be discussed in this work will be cyclohexanone monooxygenase CHMO). Also from the same biodegradation pathway as CHAO this enzyme is responsible for the stereo- and regio-specific conversion of small cyclic ketones into lactones. This reaction, known as the Baeyer-Villiger Oxidation, is of tremendous importance in the pharmaceutical industry as lactones often serve as the building blocks of other larger compounds. Surprisingly, despite many years of research there has never been a published report on the structure of this enzyme. We detail here, for the first time, the crystallographic structure of CHMO in multiple conformations. Together, the various structures of CHAO and CHMO presented will provide insight into how bacteria are able to process xenobiotics and how we might use this information for structure based protein design.

Naturally occurring alkaloid, 10-hydroxy-camptothecin 10HC) is a promising structural derivative of camptothecin which possesses the ability to inhibit a wide range of human tumors. However, its anti-tumor potential has not been fully realized owing to the pH dependent hydrolytic instability and poor aqueous solubility. To obtain the kinetic and thermodynamic parameters of hydrolysis, a derivative spectrophotometric technique was used for the simultaneous estimation of lactone and carboxylate forms of 10HC. Validation of the analytical method was done with respect to reproducibility, % recovery, and level of detection. Hydrolysis of the lactone ring of 10HC followed a 1st order decay with a rate constant equal to 0.0281 ＋/- 0.001) min-1 in PBS at pH 7.4 and at a temperature of 37 &deg；C. The activation energy for the hydrolysis reaction as calculated from the Arrhenius equation was 79.41 ＋/- 0.92) kJ mol -1, whereas the enthalpy and entropy of hydrolysis of 10-hydroxy-camptothecin were on average 12.45 kJ mol-1 and 52.37 J K-1 mol-1, respectively. The positive enthalpy and entropy values of the 10HC-lactone hydrolysis indicate that the reaction is endothermic and entropically driven. Physicochemical characterization was carried out to fully characterize the poor aqueous solubility and solid-state properties of 10-hydroxy-camptothecin 10HC). Molecular and system properties were determined from titration, partition and solubility studies using UV and fluorescence spectroscopy, while solid state characterization of the 10HC was carried out with x-ray, DSC and TGA. The enthalpies of solution of the unionized and ionized forms of 10HC, as deduced from isothermal and iso-pH equilibrium solubility measurements, were 45.4 kJ&middot；mol-1 and 22.7 kJ&middot；mol-1, respectively. The pKa of 10HC was determined to be 1.42 at 25 &deg；C, while the basicity of the quinoline group of 10HC was shown to decrease with increasing temperature due to a positive enthalpy of deprotonation of 23.6 kJ.mol-1. The intrinsic partition coefficient of 10HC was determined to be 6.49, which is significantly smaller than that of the parent camptothecin. Evidently, the hydroxyl substitution on the A ring of camptothecin renders the molecule considerably more polar, though still hydrophobic and sparingly soluble in aqueous media. Dissolution studies supported by x-ray and thermal analysis revealed polymorphism and serious metastability of the 10HC anhydrous form in aqueous solutions. The aqueous solubility of 10HC-lactone monohydrate was found to be pH and temperature dependent with an estimated intrinsic solubility of 1.81 ＋/- 0.215 muM. Contrary to the low intrinsic solubility, the solubility of 10HC in extremely acidic media increased by more than 3 orders of magnitude. Furthermore, the purpose of the present work was to study the pH-solubilization behavior of 10HC, and to systematically discern the improved solubility and stability of 10-hydroxy-camptothecin in presence of pharmaceutically acceptable co-solvents and surfactants. Drug concentrations following equilibration of 10HC in various pH solutions and at different weight fractions of the cosolvents； PEG400, PEG1450, PEG6000, PEG8000, glycerol and propylene glycol and, surfactants； F-68, F127, Gelucire and Sodium Dodecyl Sulfate SDS) were determined using HPLC. The kinetic and thermodynamic parameters of hydrolysis were determined by monitoring the drug hydrolysis using a derivative spectroscopic method. Association of drug to micelles was studied using fluorescence anisotropic technique. Based on the pH-solubility profile, the pKa of the phenol group of 10HC was determined to be 7.6 ＋/- 0.023. The presence of 10HC-lactone at highly basic pH was attributed to the pseudo- equilibrium between the anionic 10HC-lactone and 10HC-carboxylate. An exponential and a linear increase in the solubility of 10HC was observed with increasing concentrations of the cosolvents and the surfactants, respectively, with PEG400 and SDS showing the maximum solubilization efficiency. Abstract shortened by UMI.)