Cytochrome P450 monooxygenases or CYPs comprise a large family of enzymes that are found in all classes of living organisms, from bacteria to man. These enzymes are involved in the metabolism of many endogenous and xenobiotic (foreign) compounds. In insects, CYPs confer resistance to various insecticides, and resistance-associated overexpression of multiple CYP genes in resistant insects is a common phenomenon. In Drosophila, multiple Cyp genes including Cyp6a2 and Cyp6a8 show higher level of expression in resistant strains than in the susceptible ones. To date, molecular basis of CYP gene overexpression has not been examined in detail. Barbiturate compounds such as phenobarbital and barbital induce both these genes. An unpublished observation from our laboratory showed that Cyp6a2 as well as Cyp6a8 are induced by over-the-counter caffeine tablet, Vivarin. In the present study, I used pure caffeine as a tool to better understand the mechanism of Cyp6a2 and Cyp6a8 gene regulation in Drosophila. The specific objectives of this project has been to (1) map the upstream DNA of both genes for the sequences responsible for caffeine-induction; (2) examine whether adenosine receptors and/or cAMP-specific phosphodiesterase (cPDE) are involved in transduction of caffeine signal to induce the two Cyp6 genes; and (3) investigate whether Drosophila AP-l transcription factors (D-JUN and D-FOS) playa role in caffeine induction of Cyp6a8 gene. For these objectives, several Cyp-luc reporter plasmids carrying firefly lueiferase (luc) reporter gene under the control of different regions of Cyp6a8 and Cyp6a2 upstream DNAs were constructed to transfect Drosophila embryonic cells, Schneider line 2 (SL-2). Two transgenic reporter strains carrying firefly lueiferase (luc) reporter gene under the control of O.8-kb or 0.2-kb upstream DNA of Cyp6a8 gene were also used to study the mechanism of caffeine induction in vivo. Results of Northern blot analysis showed that caffeine induces endogenous Cyp6a2 and Cyp6a8 genes at the steady-state mRNA levels both in reporter transgenic and wild-type flies. Transfection experiments with SL-2 cells showed that -983/-1 and 7611-11 upstream DNAs of Cyp6a2 and Cyp6a8, respectively, have sequences for caffeine-induced expression. Further transfection experiments with reporter plasmids carrying luc reporter gene attached to truncated upstream DNAs of Cyp6a2 and Cyp6a8 genes showed that the regions between -265/-129 of Cyp6a2 and -199/-109 of Cyp6a8 have sequences that confer caffeine-induced expression. However, the level of both constitutive and induced expression was highest with -981/-1 DNA of Cyp6a2and -761111 DNA of Cyp6a8genes. Sequence analysis identified several putative binding sites for Activator Protein -1 (AP-l) and cyclic AMP response element CRE binding protein. (CREB) motifs in the upstream DNA of both genes. Moreover, when the four core bases of the single AP-l site present in the -109/-11 DNA of Cyp6a8 were mutated, constitutive expression decreased by 8-fold, suggesting the positive role of AP-l in Cyp6a8 gene expression. To examine whether caffeine signaling is mediated via adenosine receptor (AdoR) and/or via cPDE inhibition, SL-2 cells transfected with Cyp6a2 and Cyp6a8 reporter constructs were treated with AdoR agonists or with antagonists or with cPDE inhibitors. The Cyp6a8-luc reporter transgenic lines were also treated with these chemicals. The results showed that caffeine signaling is mediated by PDE inhibition and via increase in the intracellular cAMP level. Indeed, treatment with dibutyryl cAMP induces Cyp6a2 as well as Cyp6a8 promoters. Since induction of cAMP pathway is known to upregulate AP-I transcription factors, effect of overexpression of Drosophila D-FOS and (or) DJUN (components of AP-I) on Cyp6a8 promoter activity in SL-2 cells was examined. Surprisingly, activity of Cyp6a8-1uc reporter construct was inhibited when D-FOS or DJUN proteins were overexpressed, suggesting that AP-I proteins are inhibitory for Cyp6 gene expression. In contrast, the Cyp6a8 promoter activity was upregulated, when cells were transfected with anti-D-JUN plasmid or when cells transfected with D-JUN or DFOS sense construct were treated with caffeine. When relation between caffeine and the two AP-I proteins was examined, it was found that caffeine treatment significantly lowers the D-JUN protein level both in SL-2 cells as well as in adult flies. Reporter gene assays and Northern blot analysis showed that caffeine treatment has no effect on the transcriptional activity of the D-jun and D-fos genes. Taken together, it might be concluded that caffeine induction of Cyp6a2 and Cyp6a8 genes is mediated via degradation of D-JUN that acts as a repressor for the promoter of Cyp6a8. Induction of the cAMP pathway and subsequent phosphorylation of the AP-l proteins may relieve the AP-l mediated-repression by promoting the degradation of these proteins. Further investigation is required to resolve these possibilities.

Sufficient pools of deoxyribonucleotide triphophates (dNTPs) are essential for the high fidelity replication and repair of DNA, the hereditary material for a majority of living organisms. Ribonucleotide reductase (Rnr) catalyzes the rate-limiting step of de-novo DNA synthesis, the reduction of ribonucleosides to deoxyribonucleosides. Since the cell relies primarily upon ribonucleotide reductase for its dNTPs, both the cellular levels and activity of Rnr are heavily regulated, especially when DNA damage occurs or during replication blocks in the cell cycle. If dNTP pools become too high, too low, or imbalanced, genomic instability results, leading to either the formation of cancerous cells or cell death. High levels of dNTPs are required by actively propagating cells for the replication of new DNA molecules. Therefore, Rnr makes an excellent target for anti-cancer, anti-microbial, and anti-fungal chemotherapeutic agents. Deficiencies in the cellular mismatch repair (MMR) machinery have been linked to genetic instability and carcinogenesis. Two alleles of Rnr1 were recently discovered, Rnr1S269P and Rnr1S610F, which have a mismatch repair synthetic lethal (msl) phenotype in Saccharomyces cerevisiae cells with missing or defective MMR genes. To uncover the molecular mechanism of the msl phenotype in these two mutants, recombinant Rnr1p-S269P and Rnr1p-S610F were subjected to in vitro activity assays, X-ray crystallography, and in vitro nucleoside-binding assays (Chapter 3). The Rnr1S269P allele was shown to dysregulate specificity cross talk by X-ray crystallography experiments, leading to reduced levels of dATP in the cell. A 2-fold reduction in binding of ADP substrate was observed in the Rnr1S610F allele, however reduction of the kcat is believed to cause the observed msl phenotype in this mutant. The first X-ray crystal structures of the large subunit of ribonucleotide reductase from Homo sapiens (hRRM1) are also presented here (Chapter 4). The hRRM1●TTP and hRRM1●TTP●GDP structures describe the binding of effector and substrate to the specificity and catalytic sites. In addition, the two structures hRRM1●TTP●ATP and hRRM1●TTP●dATP are the first X-ray crystal structures of Rnr from any species with the allosteric activity site occupied with the natural ligands ATP and dATP. Size exclusion chromatography data and a low resolution X-ray crystal structure of hexameric S. cerevisiae Rnr provide a model for dATP-dependent oligomerization.

Sister chromatid cohesion is essential for proper chromosome segregation during meiosis. However, the mechanism of meiotic cohesion in Drosophila is unclear. We describe a novel protein, SOLO (Sisters On the LOose) that is essential for meiotic cohesion in Drosophila melanogaster. solo mutations cause high nondisjunction of sister and homologous chromatids of sex chromosomes and autosomes in both sexes. In solo males, sister chromatids separate prematurely and segregate randomly during meiosis II. Although bivalents appear intact throughout meiosis I, sister centromeres lose cohesion prior to prometaphase I and orient nearly randomly on the meiosis I spindle. Centromeric foci of SMC1 are absent in solo males at all meiotic stages. SOLO and the cohesin protein SMC1 co-localize to meiotic centromeres from early prophase I until anaphase II in wild-type males but both proteins are removed prematurely from centromeres at anaphase I in mei-S332 mutants, coincident with premature loss of cohesion in those mutants. solo mutations in females cause reduced frequency of homologous recombination between X chromosomes and autosomes, partially due to the loss of inhibition of sister chromatid exchange. Synaptonemal complex assembly is severely disrupted in early meiotic stage in solo females. SOLO colocalizes with SMC1 and C(3)G in meiosis. Additionally, SOLO is required for stabilizing chiasmata generated from residual recombination events. The data about the phenotypes of solo males and females and colocalization patterns of SOLO strongly suggest SOLO is a component of potential cohesin in Drosophila meiosis. Drosophila males undergo meiosis without recombination. However, the underlying mechanism is not known. Mutations of vasa cause high frequency of X-Y exchange in meiosis. Chromatin bridges at anaphase I and II, due to dicentric recombination events, were observed in vasa males. vas and solo double mutant showed precocious segregation of homologs at metaphase I besides chromatin bridge at anaphase I and II. Our data thus for the first time demonstrate that inhibition of meiotic recombination during male meiosis requires vas function and interactions between vas and solo regulate chromosome dynamics in male meiosis.

R67 dihydrofolate reductase (DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate (DHF) to produce tetrahydrofolate (THF). The enzyme is a homotetramer and its 222 symmetry allows for binding of both ligands to a single active site pore. A productive ternary complex is formed by the binding of one molecule of DHF and NADPH and inter-ligand cooperativity has been suggested to be essential for binding and catalysis. To gain further insight into the thermodynamics involved in the ground state and the transition state, temperature dependent studies on DHF binding and catalysis were performed. It was observed that binding of both NADPH and DHF is enthalpy driven. From van’t Hoff plots, the change in enthalpy, entropy and free energy for NADPH binding to R67 DHFR in the ground state were determined. Similarly, the thermodynamics of DHF binding to the R67 DHFR-NADPH complex in the ground state were determined. Arrhenius plots were also employed to study the energetics of the transition state. A comparison of TdeltaS values (for DHF binding to R67 DHFR-NADPH complex) in both ground state and transition state indicates that TdeltaS is more negative in the transition state (–11.3 kcal/mol) as compared to the ground state (–5.4 kcal/mol). This indicates a reorientation of the substrate in the transition state. The role of water in DHF and NADPH binding to R67 DHFR was also investigated. For this, the effect of osmotic pressure on the Ka /Km of ligand binding, as well as the kcat of the reaction was studied. It was observed that the kcat of the reaction was not significantly affected, while the binding of ligands was affected with increasing osmolality. Specifically, binding of NADPH tightened as osmolality increased, while binding of DHF weakened with increasing osmolality, suggesting release of water upon NADPH binding and an uptake of water on DHF binding. Results from in vivo experiments on E.coli cells containing wild type and mutant clones of R67 DHFR were also consistent with in vitro experiments, suggesting that water is involved in ligand binding to R67 DHFR.

The dynamic nature of proteins in solution is often an indispensable factor in biological function such as enzymatic catalysis. Complementary to the conventional structural analysis, computational simulations have the advantage to reflect the dynamic nature of proteins or enzymes. One of the computational simulation methods, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations, has been widely applied to the research in structural analysis, ligand-receptor binding and enzymatic catalysis. In this dissertation, QM/MM MD simulations were applied to the studies on cytidine deaminase (CDA), yeast cytosine deaminase (yCD), and kumamolisin-As, as well as two protein lysine methyltransferases (PKMTs), DIM-5 and SET7/9. In the simulations of the transition state analogue (TSA) binding of zebularine 3, 4-hydrate to CDA and of 4-[R]-hydroxyl-3,4-dihydropyrimidine (DHP) to yCD, proton transfers were observed between the TSA and a catalytic Glu residue in both cases. Such general acidbase mechanism was also observed in the stabilization of the tetrahedral intermediate by a critical Asp residue during the acylation of kumamolisin-As. Moreover, dynamic substrate-assisted catalysis (DSAC) involving the His of the substrate at P1 site was proposed. It was suggested that DSAC may contribute to the transition state stabilizations and substrate specificity of kumamolisin-As. The origin of the product specificities of PKMTs was studied by comparison of QM/MM MD simulations on the first, second and third methyl transfers in the trimethylase DIM5 and the monomethylase SET7/9. The product specificities of the enzymes can be well explained by population distributions of well-aligned reactive structures and the relative free energy barriers for the methyl transfers. The structural and energetic reasons for the product specificities were discussed and a triplet code based on the relative free energy barriers for the three methyl transfers was proposed in the determination of product specificities of PKMTs.

A cDNA was isolated from soybean (Glycine max) nodules that encodes a putative transporter (GmN70). GmN70 is expressed predominantly in mature nitrogenfixing root nodules. By western-blot and immunocytochemical analyses, GmN70 was localized to the symbiosome membrane of infected root nodule cells, suggesting a transport role in symbiosis. To investigate its transport function, cRNA encoding GmN70 was expressed in Xenopus laevis oocytes, and two-electrode voltage clamp analysis was performed. Ooctyes expressing GmN70 showed outward currents that are carried by anions with a selectivity of nitrate > nitrite >> chloride. These currents showed little sensitivity to pH or the nature of the counter cation in the oocyte bath solution. One-half maximal currents were induced by nitrate concentrations between 1 to 3 mM. A global protein BLAST search for NLAT-related proteins revealed the presence of multiple homologs in all plant genomes sequenced with members segregating into 6 distinct monophyletic clades. Two genes from Arabidopsis clustering within the same clade 1 (chromosome nomenclature:At2g39210 and At2g28120) and shared a high amino acid sequence identity with GmN70 (respective 63.7% and 56.3%) and cluster in the same clade (Clade 1) as GmN70 and LjN70. Investigation of the transport properties of these two Arabidopsis NLAT-like genes (renamed AtNLAT1;1 [At2g39210] and AtNLAT1;2 [At2g28120] showed similar transport properties as GmN70 and LjN70 and transported the inorganic anions, nitrate, nitrite, and chloride. Expression analysis and sub-cellular localization of AtNLAT1;1 show it to be expressed predominately in the leaf on the plasma membrane. Its expression is regulated by a diverse set of biotic and abiotic stress signals including Pseudomonas syringae pv. tomato, salicylic acid, salinity, touch, and wounding suggesting a role in stress adaptation in plants. Consistent with these findings, transgenic Arabidopsis lines in which AtNLAT1;1 expression is knocked-down show an enhanced sensitivity to NaCl as well as a perturbed ionomic profile as revealed by inductively coupled plasma-mass spectroscopy (ICP-MS). Overall, the data presented in this body of work have provided insight into the biochemical and biophysical function and expression profiles of two members of the NLAT family, GmN70 and AtNLAT1;1.

The majority of chloroplast proteins are nuclear encoded and transcribed on cytosolic ribosomes, and therefore must be post-translationally imported into the chloroplast. Preproteins are directed to the chloroplast via a cleavable Nterminal extension known as a transit peptide. This transport is mediated by the Toc and Tic complexes (Translocon at the Outer/Inner Chloroplast envelope membrane), functioning in tandem to transport preproteins into chloroplasts relying on the hydrolysis of ATP and GTP. The Toc complex is composed of the β-barrel channel protein Toc75 and the homologous GTPase receptors Toc34 and Toc159. GTP hydrolysis is necessary for the formation of the early import intermediate, in which the transit peptide is inserted into the Toc channel, but the presence of internal ATP is the only energetic requirement for the later stages of translocation to occur, mediated by the stromal motor complex with an Hsp100 isoform hydrolyzing stromal ATP. The purpose of the current study is to characterize the change in stability and/or oligomeric status of the Toc complex with the incubation of nucleotides, analogs, proteins/peptides, etc. by blue native electrophoresis followed by 2d SDS-PAGE. The Toc complex ranges from ~800 kDa to greater than 1320 kDa for the proposed Toc/Tic supercomplex when no proteolytic degradation has occurred. Proteolytic degradation of Toc159 is correlated with the appearance of complexes with a mass ranging from 800 kDa to 440 kDa and below. Proteolytic degradation of Toc159 is more apparent in chloroplasts purified from older Pisum sativum plants. The results of the incubation of chloroplasts with GDP, GTP, and non-hydrolyzable analogs before analysis by 2d electrophoresis followed by western blot hybridization suggest that the loading of the GTPase receptors with nucleotide triphosphate results in the increased association of Toc components in complexes in the size range of 880- 630 kDa.

Glutamine synthetase (GS) plays an important role in nitrogen metabolism, where it catalyzes the ATP-dependent condensation of glutamate and ammonia to yield glutamine. Recent studies showed the importance of M. tuberculosis GS (MtGS) for growth and survival of the bacterium, and demonstrated its potential as a drug target. This thesis presents structural studies of MtGS and mammalian GSs, which are aimed at identifying and developing novel inhibitors against the mycobacterial target. The structure of MtGS was solved in complex with a phosphorylated form of the inhibitor methionine sulfoximine, magnesium and ADP. The complex structure provides a detailed picture of the active site, offering several insights into catalysis and inhibition, as well as forming a solid basis for structure-based drug design. The apo canine GS and liganded human GS structures described in this thesis represent the first structures of the mammalian enzymes. Comparison of the structures revealed substrate-induced conformational changes. Inspection of the nucleotide-binding site showed that it differs from that of MtGS, thus offering good opportunities to design specific and selective inhibitors of the mycobacterial enzyme. The amino acid-binding site of MtGS was evaluated as a target for inhibition, using a combination of a literature survey, structure-based virtual screening and the synthesis of a small library of compounds. As a result, several new inhibitors of MtGS could be identified. Finally, the structural basis for inhibition of MtGS by a purine analogue (PA) is provided. PA, an analogue of a class of compounds found to inhibit MtGS in a high-throughput screening assay, targets the nucleotide-binding site. The architecture of the HsGS nucleotide-binding site indicates that PA would not be able to bind to the human enzyme, offering good prospects for selective inhibition of MtGS.

The actin cytoskeleton is a complex structure that performs a wide range of cellular functions including: cell locomotion, cytokinesis, chemotaxis, signal transduction and apoptosis. The coordinated assembly and disassembly of actin filaments is controlled by a multitude of proteins (ABPs) in the cell. There are over 160 actin-binding proteins known, which with actin, account for approximately 25% of cellular protein. ABPs are classified to several major groups based on their sequence identity and functions. In this work, we have elucidated the crystal structure of ATP bound gelsolin. We have shown that ATP binding involves the two halves of gelsolin through forming numerous polar and hydrophobic contacts. Amino acid residues that form the ATP-binding sites in inactive gelsolin are widely dispersed in the activated molecule, and hence, ATP binding is disrupted on gelsolin activation. This suggests that binding of ATP may modulate the sensitivity of gelsolin to calcium ions. The structure of human gelsolin domains 1-3 bound to actin revealed a calcium ion bound to domain 2. Here, we demonstrated that only two calcium ions are needed to activate geloslin. We speculate that this domain 2 calcium ion and the one in domain 6 participate in the initial activation of gelsolin. The crystal structure of the activated adseverin C-terminus is highly similar to that of the C-terminus of gelsolin. Comparative analysis suggests that, like the gelsolin C-terminus, adseverin will also contact actin through domain 4 and domain 6. Biochemical experiments, presented here, show that a minimum of one calcium is required for adseverin to depolymerizing actin filaments compared to two calcium for gelsolin. We speculate that this is due to the lack of the C-terminal extension in adseverin. We undertook a comparative analysis of four members of the gelsolin family proteins, gelsolin, adseverin, villin and capG, in the aspects of their calcium binding, pH activation and ATP binding. The results show that only gelsolin and adseverin are able to depolymerize actin filaments at pH < 6 in the absence of calcium ions and only gelsolin bind to ATP.

Biomineralization, one of the most widespread processes in nature, uses polyanionic proteins to direct oriented crystal growth. In bone and dentin, this process is under precise control of the collagen template and the noncollagenous acidic phosphoproteins. These phosphoproteins function differently depending on their sizes and level of phosphorylation. The goal of this research is to investigate the in vitro phosphorylation as well as the phosphorylation in mammalian cells of two highly phosphorylated bone/dentin extracellular matrix proteins: dentin phosphophoryn (DPP) and dentin matrix protein 1 (DMP1). This data will be important to the general hypothesis, that the phosphorylation of non-collagenous proteins play a significant role in matrix mediated mineralization. Our data shows that the in vitro phosphorylation of DPP and DMP1 could be optimized by adjusting the phosphorylation reaction time, calcium concentration, and protein modification by assessing various forms (with or without the C or N terminal end). Following the in vitro phosphorylation, mass spectrometry analysis was used to identify the sites of phoshorylation. In addition, to identify the kinases involved in phosphorylating DMP1, cell lysates from cells that have (MC3T3) and do not have (NIH3T3) the ability to mineralize their matrix and were isolated and analyzed by zymogram. Casein kinase II catalytic subunit was identified in addition to potential novel kinases responsible for DMP1 phosphorylation. The second goal of this research is to assess if cells that have the ability to form a mineralized matrix will possess specialized kinases that can phosporylate these highly phosphorylated and acidic proteins. To achieve this goal we over-expressed and purified DMP1 from two cell types: 1) cells that have the ability to mineralize their matrix and 2) cells that do not possess the ability to mineralize their matrix. The purified proteins were then analyzed by SDS-PAGE and mass spectrometry to quantify and determine the sites of phoshorylation. This study has expanded our knowledge on the mechanisms involved in the phosphorylation of DPP and DMP1 and provided the parameters to start assessing the role of phosphorylation on tissue mineralization.