Campylobacter jejuni is one of the leading bacterial causes of human gastroenteritis world wide and has been linked to several severe complications including autoimmune syndromes which can result in paralysis. Despite being the subject of much study, C. jejuni remains a major public health burden in both developing and developed nations. There is currently no vaccine available for protection against this pathogen and the mechanisms important for C. jejuni pathogenesis are not fully defined. This study has employed a range of experimental approaches to investigate the molecular mechanisms involved in C. jejuni pathogenesis. Lipooligosaccharides (LOSs) are surface structures and known virulence factors of C. jejuni which are involved in serum resistance, resistance to phagocytic killing, endotoxicity and adhesion. Mutagenesis studies targeting the putative LOS biosynthesis genes wlaRF, wlaTA, wlaTB, wlaTC and waaV were performed in order to characterise the proteins encoded by each of these six genes and assess their potential role in C. jejuni pathogenesis in vitro. The gene product of wlaTA was found to be essential for C. jejuni survival and therefore a knock out mutant could not be generated. Phenotypic characterisation of four knock-out mutants confirmed that each gene contributed to the construction of the LOS molecule as all four mutants produced a truncated LOS moiety and altered their immunoreactivity. Further analysis determined that the production of complete LOSs was important for C. jejuni to invade and adhere to both human and chicken cells in vitro. This study identified a link between the inactivation of two LOS biosynthesis genes and the loss of motility, another important virulence factor. A major source of human C. jejuni infection is contact with contaminated poultry. However, C. jejuni exists as a commensal in chickens. It is currently not known why C. jejuni is pathogenic to humans and not to chickens and the differences between these two hosts represent pathogenic and non-pathogenic environments respectively. These environmental differences were exploited in this study. The four conditions investigated were temperature, blood, bile and host cells in vitro. Five different C. jejuni strains (NCTC11168, 81116, HB93-13, a recent human enteritis isolate and a recent chicken isolate) were subjected to modelled

In recent years, the methyl erythritol phosphate (MEP) pathway to isoprenoids has been the subject of intensive research. The interest is because isoprenoids have important roles in many cellular processes essential for the survival of several pathogenic organisms, making the inhibition of this pathway an attractive target for the drug discovery. The second enzyme in the MEP pathway is 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). DXR is a promising target for the development of new antibiotics, antimalarials and herbicides. The overall objective of this research was a better understanding of DXR by using site-directed mutagenesis guided by crystal structure analysis and inhibition studies. One set of mutants was designed to expand the selectivity of DXR. An analog of DXP, 1,2-dideoxy-D-threo-3-hexulose 6-phosphate (1-methyl-DXP or Me-DXP), that differs from DXP by having an ethyl ketone, rather than a methyl ketone, was reported to be a weak competitive inhibitor. Using the x-ray crystal structures of DXR as a guide, a highly conserved tryptophan residue in the flexible loop was identified as a potential steric block to the use of this analog as a substrate. Four mutants of Synechocystis sp. PCC6803 DXR, named W204F, W204L, W204V and W204A, were prepared and characterized. The W204F mutant was found to utilize the analog Me-DXP as a substrate. The roles of amino acids residues shown to be in the DXR active site in the available E. coli crystal structures were also studied. Mutants at the positions Dl52, S153, E154, H155, M206 and E233, were prepared. The kinetic characterization of these mutants showed that the amino acid substitution, conservative or not, in these residues reduced the DXR catalytic activity, confirming that these are key amino acids responsible for the DXR catalytic efficiency. Inhibition studies of the E. coli DXR by fosmidomycin in the presence of Co²⁺, Mg²⁺ and Mn²⁺ showed that this inhibition is not dependent on a specific divalent cation. Inhibition of the Synechocystis sp. PCC6803 DXR by fosmidomycin and its hydroxamate and FR 900098 analogs was conducted showing that these compound are potent inhibitors of this enzyme. Fosmidomycin and FR900098 have inhibition constants in the low nM range. In addition the patterns of the progress curves for fosmidomycin, its hydroxamate analog and FR900098 were shown to be prototypical for slow, tight-binding inhibitors, as was seen for these inhibitors with the E. coli enzyme. / Graduation date: 2005

Graduation date: 2000

Carbon Monoxide Dehydrogenase (CODH), also known as Acetyl-CoA synthase (ACS), is one of seven known Ni containing enzymes. CODH/ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl-CoA. Recently, X-ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr) and CODH from Carboxydothermus hydrogenoformans (CODHCh) have been published. These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the β subunit of heterotetrameric α2β2 enzyme from Moorella thermoacetica (CODHMt), formerly Clostridium thermoaceticum. Neither CODHRr nor CODHCh contain an α-subunit as is found in CODHMt. The precise structure of the active site for acetyl-CoA synthase, called the A-cluster, is not known. Therefore, crystallization of the α subunit is required to solve the remaining structural features of CODH/ACS. Obtaining crystals and determining the X-ray crystal structure is a high-risk endeavor, and a second project was pursued involving the preparation, expression and analysis of various site-directed mutants of CODHMt. Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed. Visual inspection of the crystal structure of CODHRr and CODHCh, along with sequence alignments, indicates that there may be separate pathways for proton and electron transfer during catalysis. Mutants of a proposed proton transfer pathway were characterized. Four semi-conserved histidine residues were individually mutated to alanine. Two (His116Mt and His122Mt) were essential to catalysis, while the other two (His113Mt and His119Mt) attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway.

A major obstacle for developing new antidepressants has been limited knowledge of the structure and function of a central target, the serotonin transporter (SERT). Established SERT inhibitors (SSRIs) were docked to an in silico SERT model to identify likely binding pocket amino acid residues. When mutated singly, no one of five implicated residues was critical for high affinity in vitro binding of SSRIs or cocaine. The in silico SERT model was used in ligand virtual screening (VS) of a small molecule structural library. Selected VS "hit" compounds were procured and tested in vitro; encouragingly, two compounds with novel structural scaffolds bound SERT with modest affinity. The combination of computational modeling, site-directed mutagenesis and pharmacologic characterization can accelerate binding site elucidation and the search for novel lead compounds. Such compounds may be tailored for improved serotonin receptor selectivity and reduced affinity for extraneous targets, providing superior antidepressants with fewer adverse effects. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmacology / MS; / Thesis;

Inositol dehydrogenase (IDH, EC 1.1.1.18) from <i>Bacillus subtilis</i> catalyzes the reversible NAD⁺-dependent oxidation of the axial hydroxyl group of <i>myo</i>-inositol to form 2-keto-<i>myo</i>-inositol, NADH and H⁺. IDH is the first enzyme in catabolism of myo-inositol, and <i>Bacillus subtilis</i> is able to grow on <i>myo</i>-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of <i>myo</i>-inositol.<p> In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the <i>apo</i>-IDH, <i>holo</i>-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD⁺-dependent to 6-fold preference for NADP⁺ over NAD⁺ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed <i>myo</i>-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.

MenD, a thiamin diphosphate (ThDP)-dependent enzyme, catalyzes the reaction from isochorismate (ISC) to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and thus is also called SEPHCHC synthase. This conversion is the first committed step in the classical menaquinone (Vitamin K2) biosynthetic pathway, requiring 2-ketoglutarate (2-KG), ThDP and Mg²⁺. Since the biosynthesis of menaquinone is essential in some bacterial pathogens, for example <i>Mycobacterium tuberculosis</i>, MenD or the menaquinone pathway could be a target for drug development.<p> The method for the kinetic assay of the MenD-catalyzed reaction was evaluated by comparing UV spectrophotomeric measurements and HPLC analysis. It was validated that the steady-state kinetics of the MenD-catalyzed reaction can be determined by monitoring UV absorbance of ISC at 278 nm and 300 nm.<p> Phosphonate analogues of 2-KG were synthesized and assayed as inhibitors of the MenD reaction. It was found that the phosphonate analogues of 2-KG are competitive inhibitors with varied affinity for MenD. Of the inhibitors, monomethyl succinyl phosphonate (MMSP) was the most effective, with a <i>K</i>_i of 700 nM. However, the potent MenD inhibitors show no effectiveness against mycobacterial growth.<p> An analogue of isochorismate, trans-(±)-5-carboxymethoxy-6-hydroxy-1,3-cyclohexadiene-1-carboxylate ((±)-CHCD), was synthesized. The (+)-CHCD was found to be an alternative substrate for the MenD-catalyzed reaction. When CHCD was utilized in the MenD reaction, 5-carboxymethoxy-2-(3-carboxy-propionyl)-6-hydroxy-cyclohex-2-enecarboxylate (CCHC) was isolated and characterized, which was believed to be the product of spontaneous isomerization of the SEPHCHC-like analogue. The kinetic study of MenD reaction using (±)-CHCD, in association with the kinetics pattern probed by MMSP, demonstrated for the first time that the MenD-catalyzed reaction has a Ping Pong bi bi kinetic mechanism.<p> The analysis of sequence and structure of MenD from E. coli allowed the investigation of the active site residues and their catalytic functions by mutation of the individual residues. S32A, S32D, R33K, R33Q, E55D, R107K, Q118E, K292Q, R293K, S391A, R395A, R395K, R413K and I418L were prepared and assayed kinetically with respect to 2-KG, ISC, (±)-CHCD, ThDP and Mg²⁺. The values of <i>K</i>_m^a and <i>k</i>_cat^a/<i>K</i>_m^a for the mutants, in comparison with that of wild type MenD, provide valuable insight into the catalytic mechanism of MenD.

MenD, a thiamin diphosphate (ThDP)-dependent enzyme, catalyzes the reaction from isochorismate (ISC) to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and thus is also called SEPHCHC synthase. This conversion is the first committed step in the classical menaquinone (Vitamin K2) biosynthetic pathway, requiring 2-ketoglutarate (2-KG), ThDP and Mg²⁺. Since the biosynthesis of menaquinone is essential in some bacterial pathogens, for example <i>Mycobacterium tuberculosis</i>, MenD or the menaquinone pathway could be a target for drug development.<p> The method for the kinetic assay of the MenD-catalyzed reaction was evaluated by comparing UV spectrophotomeric measurements and HPLC analysis. It was validated that the steady-state kinetics of the MenD-catalyzed reaction can be determined by monitoring UV absorbance of ISC at 278 nm and 300 nm.<p> Phosphonate analogues of 2-KG were synthesized and assayed as inhibitors of the MenD reaction. It was found that the phosphonate analogues of 2-KG are competitive inhibitors with varied affinity for MenD. Of the inhibitors, monomethyl succinyl phosphonate (MMSP) was the most effective, with a <i>K</i>_i of 700 nM. However, the potent MenD inhibitors show no effectiveness against mycobacterial growth.<p> An analogue of isochorismate, trans-(±)-5-carboxymethoxy-6-hydroxy-1,3-cyclohexadiene-1-carboxylate ((±)-CHCD), was synthesized. The (+)-CHCD was found to be an alternative substrate for the MenD-catalyzed reaction. When CHCD was utilized in the MenD reaction, 5-carboxymethoxy-2-(3-carboxy-propionyl)-6-hydroxy-cyclohex-2-enecarboxylate (CCHC) was isolated and characterized, which was believed to be the product of spontaneous isomerization of the SEPHCHC-like analogue. The kinetic study of MenD reaction using (±)-CHCD, in association with the kinetics pattern probed by MMSP, demonstrated for the first time that the MenD-catalyzed reaction has a Ping Pong bi bi kinetic mechanism.<p> The analysis of sequence and structure of MenD from E. coli allowed the investigation of the active site residues and their catalytic functions by mutation of the individual residues. S32A, S32D, R33K, R33Q, E55D, R107K, Q118E, K292Q, R293K, S391A, R395A, R395K, R413K and I418L were prepared and assayed kinetically with respect to 2-KG, ISC, (±)-CHCD, ThDP and Mg²⁺. The values of <i>K</i>_m^a and <i>k</i>_cat^a/<i>K</i>_m^a for the mutants, in comparison with that of wild type MenD, provide valuable insight into the catalytic mechanism of MenD.

Inositol dehydrogenase (IDH, EC 1.1.1.18) from <i>Bacillus subtilis</i> catalyzes the reversible NAD⁺-dependent oxidation of the axial hydroxyl group of <i>myo</i>-inositol to form 2-keto-<i>myo</i>-inositol, NADH and H⁺. IDH is the first enzyme in catabolism of myo-inositol, and <i>Bacillus subtilis</i> is able to grow on <i>myo</i>-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of <i>myo</i>-inositol.<p> In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the <i>apo</i>-IDH, <i>holo</i>-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD⁺-dependent to 6-fold preference for NADP⁺ over NAD⁺ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed <i>myo</i>-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.

The histidine-containing protein (HPr) is a central phosphotransfer component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) that transports carbohydrates across the cell membrane of bacteria. There are two HPr phosphorylation events investigated in this thesis. Firstly, BPr from Gram-positive species may undergo a regulatory phosphorylation of an absolutely conserved Ser46 residue. There are numerous metabolic consequences to this phosphorylation, including inducer exclusion and expulsion, inhibition of PTS sugar uptake and catabolite repression. While HPr from Gram-negative sources cannot undergo phosphorylation of Ser46 'in vivo' or ' in vitro' it is possible to mimic the phosphorylation through the Ser46Asp mutation. To determine the structural consequences of the mutation the crystallographic structure of the 'E. coli'. Ser46Asp HPr was determined at 1.5 Å resolution. The structure revealed that no significant structural rearrangements are induced by the mutation and the inability to accept phosphotransfer from Enzyme I is due to electrostatic disruption of the interaction of these proteins. Phosphorylation of an absolutely conserved His15 for the purpose of phosphotransfer represents the second phosphorylation event to be investigated. The absolute requirement for histidine at the 15 position was investigated through mutagenesis. The mutation of His15Asp of 'E. coli' HPr was able to accept a phosphoryl group from Enzyme I and further transfer the phosphoryl group to Enzyme IIAglc. None of the other mutations of the fifteen position were able to be phosphorylated. The His15Asp mutant had a Vmax of 0.1% and a ten-fold increase in Kin with respect to wild type HPr. As a consequence of the phosphorylation of His15Asp HPr a third protein species of higher pI than the original protein was identified. This high pI species seemed to share numerous similarities to succinimides which are known to be involved in deamidation. The inability to detect the known degradation products of succinimides suggested that the high pI species may involve isoimide formation. Isoimides have been proposed, but never experimentally demonstrated in proteins. A mechanism through which the phosphoacyl intermediate may catalyze isoimide formation is proposed. In addition the potential involvement of isoimide formation as a mechanism in physiological regulatory signaling is discussed.