How an enzyme achieves such high rates of catalysis in comparison to its solution counterpart reaction has baffled scientists for many decades. Much of our understanding of enzyme function is derived from research devoted to enzyme chemical reactions and analysis of static three-dimensional images of individual enzyme molecules. However, more recently, a role of protein dynamics in facilitating enzyme catalysis has emerged. It is often challenging to probe how protein motions are correlated to and impact on the catalytic cycle of enzymes. Nevertheless, this subject must be addressed to further our understanding of the roots of enzyme catalysis. Herein, this research question is approached by studying the link between protein domain dynamics and electron transfer chemistry in the diflavin oxidoreductase family of enzymes. Previous studies conducted on the diflavin oxidoreductases have implied a role of protein domain dynamics in catalysing electron transfer chemistry. However, diflavin oxidoreductase motions have not been experimentally correlated with mechanistic steps in the reaction cycle. To address these shortcomings, a 'real-time' analysis of diflavin oxidoreductase domain dynamics that occur during enzyme catalysis was undertaken. The methodology involved specific labelling of diflavin oxidoreductases (cytochrome P450 reductase, CPR, and neuronal nitric oxide synthase, nNOS) with external donor-acceptor fluorophores that were further used for time-resolved stopped-flow Förster resonance energy transfer (FRET) spectroscopy measurements. This approach to study enzyme dynamics was further linked with traditional UV-visible stopped-flow approaches that probed enzymatic electron transfer chemistry. Results showed a tight coupling between the kinetics of electron transfer chemistry and domain dynamics in the two diflavin oxidoreductase systems studied. Moreover, through the use of a flavin analogue (5-deazaflavin mononucleotide) and isotopically labelled nicotinamide coenzymes (pro-S/R NADP2H), key steps in the reaction mechanism were correlated with dynamic events in calmodulin, the partner protein of nNOS.The approaches developed in this project should find wider application in related studies of complex electron-transfer enzymes. Altogether, this research emphasises the key link between protein domain motions and electron transfer chemistry and provides a framework to describe the relationship between domain dynamics and diflavin oxidoreductase function.

by Wong Kin Kwan Kelvin. / Thesis submitted in: September 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 91-101). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Abbreviations --- p.x / List of Figures --- p.xiii / List of Tables --- p.xiv / Chapter Chapter 1 --- Intorduction --- p.1 / Chapter 1.1 --- Alzheimer，s disease --- p.1 / Chapter 1.2 --- Histopathological features --- p.1 / Chapter 1.3 --- Tau protein pathology and AD --- p.4 / Chapter 1.4 --- Tau protein kinase I (TPKI)- GSK-3β --- p.6 / Chapter 1.5 --- Tau protein kinase II (TPKII)- Cyclin dependent kinase 5 (Cdk5) --- p.8 / Chapter 1.6 --- Available treatment --- p.9 / Chapter 1.7 --- Objectives of the present study --- p.12 / Chapter Chapter 2 --- Screening for GSK-3p inhibitors from Traditional Chinese Medicine (TCM) --- p.13 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.1.1 --- Phosphorylation of tau in AD --- p.13 / Chapter 2.1.2 --- Gsk-3p inhibitors --- p.14 / Chapter 2.1.3 --- Screening of GSK-3β inhibitor from TCM --- p.16 / Chapter 2.2 --- Material and Methods --- p.18 / Chapter 2.2.1 --- Preparation of extracts and fractions (AOF1-5) --- p.18 / Chapter 2.2.2 --- General cell culture techniques --- p.21 / Chapter 2.2.3 --- "3-(4,5-dimethyltiazoI-2-yl)-2, 5-diphenyl-tetrazolium (MTT) assay of AOF" --- p.23 / Chapter 2.2.4 --- Recombinant DNA techniques --- p.23 / Chapter 2.2.5 --- Transfection of GSK-3β and tau cDNA into COS7 cells --- p.28 / Chapter 2.2.6 --- Extraction of total proteins from culture cells --- p.28 / Chapter 2.2.7 --- Quantitation of protein by the Bradford method --- p.29 / Chapter 2.2.8 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.2.9 --- Western blot analysis --- p.31 / Chapter 2.2.10 --- GSK-3β kinase assay --- p.32 / Chapter 2.2.11 --- Determination of lithium content by atomic adsorption spectrophotometry --- p.34 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Establishment of a co-transfected cell model for GSK-3β induced tau hyperphosphorylation --- p.35 / Chapter 2.3.2 --- Preliminary screening results of aqueous and ethanol extracts (AOF1 and AOF2) --- p.37 / Chapter 2.3.3 --- Ethanol extract of AOF inhibits GSK-3p induced tau phosphorylation in COS-7 cells --- p.40 / Chapter 2.3.5 --- Effect of the essential oils of AOF on GSK-3P induced tau phosphorylation --- p.46 / Chapter 2.3.6 --- The effect of AOF essential oil on GSK-3P activity in COS7 --- p.50 / Chapter 2.3.7 --- Lithium content of AOF extracts --- p.52 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 4 --- Evaluation of the in vivo efficacy of cryptotenshinone (CT) in Morris Water Maze Task (WMT) --- p.59 / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.1.1 --- Involvement of Cholinergic system in cognitive dysfunction in AD --- p.59 / Chapter 4.1.2 --- Animal model for Alzheimer's disease --- p.60 / Chapter 4.1.3 --- Morris Watermaze Task (WMT) --- p.61 / Chapter 4.2 --- MATERIAL AND METHODS --- p.64 / Chapter 4.2.1 --- Morris Water maze setup --- p.64 / Chapter 4.2.2 --- Animal model --- p.66 / Chapter 4.2.3 --- Drug preparation --- p.67 / Chapter 4.2.4 --- Toxicity test of CT --- p.67 / Chapter 4.2.5 --- Water maze task (WMT) --- p.68 / Chapter 4.2.6 --- Visual acuity test --- p.73 / Chapter 4.3 --- RESULTS --- p.74 / Chapter 4.3.1 --- Chronic crytotanshinone treatment does not cause hepatic damages to the mice --- p.74 / Chapter 4.3.2 --- Training Session --- p.76 / Chapter 4.4 --- DISCUSSION --- p.85 / Chapter Chapter 5 --- General Discussion and Future Directions --- p.87 / Chapter 5.1 --- "AOF, the potential GSK-3 inhibitor" --- p.87 / Chapter 5.2 --- CT´ؤthe AChEI --- p.88 / References --- p.91 / Appendix --- p.102 / Chapter A1 --- Reagents for SDS-PAGE --- p.103 / Chapter A3 --- Solution components provided by QIAGEN Plasmid Maxipreps kit --- p.108 / Chapter A4 --- Reagents and medium for cell culture --- p.109 / Chapter A5 --- Reagents for kinase assay --- p.110 / Chapter A6 --- Raw data of figures --- p.112 / Chapter A7 --- Plasmid map of PCI-neo --- p.119

Further studies were done to investigate the mechanism responsible for 1-BI-induced apoptosis in NCI-H460. It was found that 1-BI stimulated the expression of pro-apoptotic p53, Bax and cytosolic NF-kB p65 subunit but decreased pERK in NCI-H460 cells. The active forms of caspase 3 and caspase 9 were detected by Western blot, accompanied by an increase in caspase 3 activity. Reactive oxygen species (ROS) was highly generated at 24 hours after the treatment and the mitochondrial membrane potential was significantly decreased at 48 and 72 hours. The application of either N-acetyl cysteine (NAC) or glutathione (GSH) attenuated the cell growth inhibition caused by 1-BI. NCI-H460 cells pretreated with NAC showed a decrease in ROS production and p65 protein but an increase in pERK. / Taken together, these findings suggest that the inhibition of THXS suppresses lung cancer cell growth by promoting either G1 cell cycle arrest or apoptosis. The status of p53 is critical for both cell cycle arrest and apoptosis in 1-BI-mediated growth inhibition, which is evident by enhanced apoptosis detected in p53-transfected NCI-H23 and DMS 114 cells and G1 cell cycle in lung cancer cells treated with PFT-alpha. The 1-BI-induced growth-inhibitory pathway is associated with the generation of ROS, alteration of mitochondrial membrane potential, down-regulation of pERK and p65. / The result showed that THXS expressed in all of the three lung cancer cell lines (NCI-H23, DMS 114 and NCI-H460). The activity of THXS was also reflected by the presence of THXS metabolite thromobxane B2 (TXB2) in the cells, which was detected by ELISA. 1-Benzylimidazole (1-BI), a specific THXS inhibitor, suppressed the lung cancer cell proliferation measured by MTT assay. 1-BI treatment caused G1 phase arrest and enhanced the level of cyclin dependent kinase inhibitor p27 in a time-dependent manner in NCI-H23 and DMS 114 cells. It markedly increased DNA fragmentation in NCI-H460 cells. The findings suggest that 1-BI inhibits cell growth by arresting cell cycle and inducing cell death. Annexin V/PI staining revealed that the cell death induced by I-BI was mainly in the format of apoptosis. Further experiments showed that the I-BI-induced apoptosis could be enhanced by the introduction of p53 into NCI-H23 and DMS 114 cells, and such enhancement was associated with a decrease in mitochondrial membrane potential. This result suggests that the p53 may play a positive role in apoptosis induced by 1-BI through changing of the mitochondrial membrane potential. The role of p53 in I-BI-mediated apoptosis was further confirmed by the experiment of the p53 inhibition. Pifithrin-alpha hydrobromide (PFT-alpha), a p53 specific inhibitor, suppressed the 1-BI-induced p53 protein expression and increased G1 cell cycle arrest. / Thromboxane A2 (TXA2) is a potent arachidonate metabolite in the cyclooxygenase-2 (COX-2) pathway, which is produced by a member of cytochrome P450 (CYP) superfamily called thromboxane synthase (THXS). Recent studies have showed that thromboxane and THXS are associated with cancer cell migration, angiogenesis, tumor metastasis and cancer proliferation but there is limited information on their role in lung cancer development. This thesis is to test the hypothesis that inhibition of THXS could alter lung cancer cell growth. / Leung, Kin Chung. / Adviser: George G. Chen. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3319. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 130-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Oxisteróis (por exemplo, 7hidroxicolesterol) são gerados por modificações oxidativas que ocorrem na molécula de colesterol. Podem ser encontrados em elevados níveis plasmáticos em pacientes com aterosclerose e como componentes da placa aterosclerótica. Considerando que o colesterol é o principal componente da cavéola (domínios específicos da membrana plasmática que ancoram diversas proteínas de sinalização) formulamos a hipótese que os oxisteróis podem ser incorporados a estes domínios, interferindo com as vias de sinalização aí localizadas. Células endoteliais de veia umbilical humana (HUVECs) em cultura foram expostas a 7hidroxicolesterol (10g/mL) por diferentes tempos. Analisamos a incorporação desse oxisterol à cavéola utilizando espectrometria de massa e a atividade das proteínas de sinalização presentes neste domínio: óxido nítrico sintase endotelial (eNOS), CD40/CD40L, receptor do fator de crescimento de fibroblastos (rFGF), utilizando PCR quantitativo e imunoblots. Inicialmente mostramos que o 7hidroxycholesterol, em concentrações fisiológicas, foi incorporado às cavéolas mais acentuadamente que em outros domínios de membrana. Esse fenômeno impediu o desligamento entre eNOS e caveolina, prejudicando a função dessa enzima. Também mostramos que o receptor CD40 apresentou uma maior incorporação à cavéola e o rFGF manteve uma ativação mais longa quando células foram expostas ao 7hidroxicolesterol. Esses efeitos gerados pelo oxisterol não estavam relacionados à sua ação sobre mediadores inflamatórios ou receptores nucleares, desde que nenhuma diferença foi observada no perfil de citocinas ou na expressão de genes dependentes da ativação de LXR. Assim, concluímos que a incorporação de 7hidroxycholesterol nos domínios de cavéola pode interferir com vias de sinalização sabidamente envolvidas na aterogênese ou na ruptura da placa / Oxysterols (for example, 7hidroxycholesterol) are generated by oxidative modifications to cholesterol molecules. They have been described in high levels in patients with atherosclerosis and as components of the atherosclerotic plaque. Since cholesterol is the main component of caveolae (plasma membrane domains that anchor several signaling proteins), we hypothesized that oxysterol could be incorporated to these domains, interfering with the signaling networks that use this pathway. Human umbilical vein endothelial cells (HUVECs) in culture were exposed to 7hidroxycholesterol (10g/mL) for different times. We analyzed incorporation of this oxysterol to caveolae using mass spectroscopy and the activity of signaling pathways present in these domains: endothelial nitric oxide synthase (eNOS), CD40/CD40L, fibroblast growth factor receptor (FGFr), using quantitative PCR and immunoblots. Initially we showed that 7hidroxycholesterol, in physiological concentrations, was incorporated to caveolae more prominently than to other plasma membrane domains. This phenomenon caused a difficulty in eNOS release from caveolin, impairing its function. We also showed that the receptor CD40 presented a stronger incorporation to caveolae and FGFr maintained a longer activation when cells were exposed to 7hidroxycholesterol. These oxysterol effects were not related to its action in inflammatory mediators or nuclear receptors, since no difference could be observed in cytokine profiles or in the expression of genes dependent on LXR activation. Therefore we conclude that 7hidroxycholesterol incorporation in caveolae domains may interfere with signaling pathways known to be involved in atherogenesis or in plaque rupture

Heterocycles play an important role in many biological processes and are widespread among natural products. Oxazole-containing natural products possess a broad range of bioactivities and are of great interest in the pharmaceutical and agrochemical industries. Herein, the biosynthetic routes to the oxazole-containing phthoxazolins and the bis(benzoxaozle) AJI9561, were investigated. Phthoxazolins A-D are a group of oxazole trienes produced by a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway in Streptomyces sp. KO-7888 and Streptomyces sp. OM-5714. The phthoxazolin pathway was used as a model to study 5-oxazole and primary amide formation in PKS-NRPS pathways. An unusually large gene cluster for phthoxazolin biosynthesis was identified from the complete genome sequence of the producer strains and various gene deletions were performed to define the minimal gene cluster. PhoxP was proposed to encode an ATP-dependent cyclodehydratase for 5-oxazole formation on an enzyme-bound N-formylglycylacyl-intermediate, and its deletion abolished phthoxazolin production. In vitro reconstitution of the early steps of phthoxazolin biosynthesis was attempted to validate the role of PhoxP, but was unsuccessful. Furthermore, Orf3515, a putative flavin-dependent monooxygenase coded by a remote gene, was proposed to hydroxylate glycine-extended polyketide-peptide chain(s) at the α-position to yield phthoxazolins with the primary amide moiety. On the other hand, an in vitro approach was employed to establish the enzymatic logic of the biosynthesis of AJI9561, a bis(benzoxazole) antibiotic isolated from Streptomyces sp. AJ9561. The AJI9561 pathway was reconstituted using the precursors 3-hydroxyanthranilic acid and 6-methylsalicylic acid and five purified enzymes previously identified from the pathway as key enzymes for benzoxazole formation, including two adenylation enzymes for precursor activation, an acyl carrier protein (ACP), a 3-oxoacyl-ACP synthase and an amidohydrolase-like cyclase. Intermediates and shunt products isolated from enzymatic reactions containing different enzyme and precursor combinations were assessed for their competence for various steps of AJI9561 biosynthesis. Further bioinformatic analysis and in silico modelling of the amidohydrolase-like cyclase shed light on the oxazole cyclisation that represents a novel catalytic function of the amidohydrolase superfamily.

Os polihidroxialcanoatos (PHA) são poliésteres bacterianos. Na sua biossíntese, a PHA sintase incorpora monômeros 3HA à cadeia polimérica. O objetivo deste trabalho foi estudar o potencial da PHA sintase de 2 isolados do gênero Burkholderia sp. na produção de copolímeros. Construiram-se linhagens recombinantes que abrigavam os genes da PHA sintase classe I, em mutantes de Pseudomonas sp. e Burkholderia sacchari, que não acumulam PHA. Foram realizados ensaios de acúmulo de PHA usando glicose como fonte de carbono, apresentando a produção de unidades de 3HB, 3HO e 3HD nas linhagens recombinantes de Pseudomonas sp. As linhagens recombinantes de B. sacchari incorporaram como único constituinte P(3HB). Ensaios de acúmulo de PHA foram realizados nas linhagens recombinantes de B. sacchari, usando como co-substratos diferentes ácidos graxos, sendo detectada a incorporação de unidades de 3HV e 3HHx além do 3HB, quando foram fornecidos acido hexanóico e valérico. Estes resultados indicam que as PHA sintases classe I são capazes de incorporar diferentes unidades monoméricas. / The polyhydroxyalkanoates (PHA) are bacterial polyester. In their biosynthesis, the PHA synthase incorporates monomers 3HA to the polymer chain. The objective of this work was to study the potential of PHA synthase of 2 isolates of the genus Burkholderia sp. in the production of copolymers. Were constructed recombinant strains that housed the genes of PHA synthase class I mutants of Pseudomonas sp. and Burkholderia sacchari, which do not accumulate PHA. PHA accumulation assays were performed using glucose as carbon source, showing the production of units of 3HB, 3HO and 3HD in recombinant strains of Pseudomonas sp. The recombinant strains of B. sacchari incorporated as single constituent P(3HB). PHA accumulation assays were performed on the recombinant strains of B. sacchari, Using as co-substrates different fatty acids, being detected the incorporation of units of 3HV and 3HHx beyond the 3HB, when were supplied hexanoic acid and valerico. These results indicate that the PHA synthases class I are able to incorporate different monomer units.

Duas enzimas têm-se destacado como possíveis marcadores biológicos periféricos na esquizofrenia: a fosfolipase A2 (PLA2) e a glicogênio sintase quinase-3 beta (GSK-3B). Essas moléculas exercem importante influência na arquitetura e plasticidade celulares, na regulação de vias metabólicas comuns (metabolismo de fosfolípides e via Wnt), em fatores de transcrição, na regulação de genes e na sobrevivência celular. Tais aspectos tornam pertinentes as investigações a respeito da possível participação dessas enzimas na fisiopatologia da esquizofrenia. Foi verificada a atividade de subtipos de PLA2 (método radioenzimático: iPLA2, cPLA2 e sPLA2) e os níveis de GSK-3B total (GSK-3Bt) e fosforilada [p(Ser9)-GSK-3B] (ELISA) em plaquetas de pacientes com esquizofrenia inicialmente livres de tratamento medicamentoso com média de 5 anos de doença (D+5a) (n=10), aos quais foi prescrita a olanzapina. Foi avaliado também um grupo de pacientes livres de tratamento medicamentoso com menos de 6 meses de sintomas psicóticos (D-6m) (n=6) aos quais foi posteriormente prescrito o haloperidol. Esses pacientes foram comparados com um grupo controle (n = 20) e avaliados longitudinalmente após o tratamento descrito por 8 semanas. Um grupo de 40 pacientes com esquizofrenia (tempo médio da doença: 17 anos) com pelo menos 6 meses de tratamento com antipsicótico (clozapina, olanzapina ou haloperidol) foi ainda avaliado. Os sintomas clínicos foram avaliados por meio da Escala de Avaliação das Síndromes Positiva e Negativa (PANSS). Quando comparado com o grupo controle, os pacientes D+5a apresentaram aumento da atividade de iPLA2 (p<0,01) e os pacientes D-6m apresentaram aumento da atividade de sPLA2 (p<0,05). Na avaliação longitudinal, somente a olanzapina diminuiu a atividade de iPLA2, cPLA2 e sPLA2 (p<0,01). Quando comparada a atividade dos subtipos de PLA2 entre os pacientes medicados a pelo menos 6 meses e o grupo controle, não foram observadas diferenças significativas. Quando comparado com o grupo controle, os pacientes D+5a apresentaram diminuição dos níveis de GSK-3Bt e p(Ser9)-GSK-3B (p<0,05). Na avaliação longitudinal, foi observado que somente a olanzapina aumentou os níveis de GSK-3Bt e p(Ser9)-GSK-3B (p < 0,01). Quando comparados os níveis de GSK-3Bt e p(Ser9)-GSK-3B entre os pacientes medicados a pelo menos 6 meses e o grupo controle não foram observadas diferenças significativas. Para os pacientes medicados a pelo menos 6 meses foram observadas correlações entre a sub-escala negativa da PANSS e os níveis de p(Ser9)-GSK-3B (r=,53, p<0,001). Sugere-se que a medicação module PLA2 e GSK-3B, independente do antipsicótico utilizado. Esses resultados apontam para uma futura aplicação dessas enzimas na verificação de adesão ao tratamento e estabilização do quadro clínico / The enzymes phospholipases A2 (PLA2) and glycogen synthase kinase-3 beta (GSK-3B) are thought to play a role in schizophrenia by influencing cellular architecture and plasticity, common signaling pathways (phospholipids metabolism and Wnt pathway), gene transcription, regulation factors and apoptosis. These aspects motivated the investigation of both these enzymes in schizophrenia. The activities of PLA2 subtypes (iPLA2, cPLA2 and sPLA2 by radio enzymatic method) and the levels of total GSK-3B and phosphorylated GSK-3B [p(Ser9)-GSK-3B] (by immune enzyme assay) were performed in platelets of drug free patients with schizophrenia for average 5 years of disease (D+5y) (n=10), who was lately prescribed with olanzapine and in drug naïve patients with less than 6 months of psychotic symptoms (D-6m) who was lately prescribed with haloperidol. These patients were compared to a control group (n=20) and were longitudinally evaluated after 8 weeks of monotherapy treatment with the prescribed antipsychotic. These enzymes were also investigated in a group of 40 patients with schizophrenia (mean duration of disease: 17 years) who were at least 6 months treated with antipsychotic (clozapine; olanzapine or haloperidol). Psychopathology was assessed with the Positive and Negative Syndrome Scale (PANSS). Patients D+5y presented higher iPLA2 activity than control group (p < 0.01) and patients D-6m presented higher sPLA2 activity than control group (p < 0.05). On longitudinal evaluation, only olanzapine decreased iPLA2, cPLA2 and sPLA2 activities (p < 0.01). In the long-term medicated patients group compared to the control group, no differences regarding PLA2 subtype activity were found. Patients D+5y presented lower GSK-3Bt and p(Ser9)-GSK-3B levels than control group (p<0.05). On longitudinal evaluation, only olanzapine increased GSK-3Bt and p(Ser9)-GSK-3B levels (p < 0.01). In the long-term medicated patients group compared to the control group, no differences regarding GSK-3B levels were found. For long-term medicated patients, it was observed correlation between p(Ser9)-GSK-3B and the PANSS negative syndrome subscale score (r = .53, p < 0.001). It was suggested that antipsychotic treatment modulated PLA2 and GSK-3B, in spite of the drug used. The results pointed to a future use of these enzymes to verify drug treatment compliance and clinical stabilization

Binding of inorganic phosphate (Pi) in ATP synthase catalytic sites is a crucial step for the synthesis of adenosine-5'-triphosphate (ATP). ATP is the fundamental means of cellular energy in almost every organism, and in order to gain insight into the regulation of ATP catalysis, critical amino acid residues responsible for binding Pi must be identified. Here, we investigate the role of highly conserved α-subunit VISIT-DG sequence residues αSer-347, αGly-351, and αThr-349 in Pi binding. Mutations αS347A/Q, αG351Q, αT349A/D/R, βR182A, and αT349R/βR182A were generated via site directed mutagenesis. Results from biochemical assays showed that αSer-347 is required for transition state stabilization and Pi binding whereas αGly-351 is only indirectly involved in Pi binding and most likely maintains structural integrity of the catalytic site. Results from preliminary experiments on αThr-349 mutants suggest that the residue may be involved in Pi binding; however, further investigation is required to fully test this hypothesis.

The soil bacterium Rhodococcus is a member of the phylum Actinobacteria and is related to Streptomyces, which is known for its production of many secondary metabolites. Recent genomic investigation of Rhodococcus has uncovered many silent gene clusters that appear to code for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKS) of unknown function. Previous work, showed that Rhodococcus species strain KCHXC3 produces an inhibitory compound in agar culture extracts that displays prominent activity against several Gram positive and Gram negative species including the pathogens Rhodococcus equi, Shigella dysenteriae and Pseudomonas aeruginosa. Using the engineered Rhodococcus transposon vector, pTNR, the goal of this investigation is to screen random mutants of KCHXC3 for strains that no longer produce the inhibitory molecule. A library of 1825 random insertion mutants was produced via electroporation then screened for production of the inhibitory molecule by a disk diffusion assay against Shigella dysenteriae. From this screening, 7 mutants which no longer produce the compound of interest were identified.

The relationship between protein structure, motions, and catalytic activity is an evolving perspective in enzymology. An interactive approach, where experimental and theoretical studies examine the same catalytic mechanism, is instrumental in addressing this issue. We combine various techniques, including steady state and pre-steady state kinetics, temperature dependence of kinetic isotope effects (KIEs), site-directed mutagenesis, X-ray crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations, to study the catalytic mechanisms of thymidylate synthase (TSase). Since TSase catalyzes the last step of the sole intracellular de novo synthesis of thymidylate (i.e. the DNA base T), it is a common target for antibiotic and anticancer drugs. The proposed catalytic mechanism for TSase comprises a series of bond cleavages and formations including activation of two C-H bonds: a rate-limiting C-H→C hydride transfer and a faster C-H→O proton transfer. This provides an excellent model system to examine the structural and dynamic effects of the enzyme on different C-H cleavage steps in the same catalyzed reaction. Our experiments found that the KIE on the hydride transfer is temperature independent while the KIE on the proton transfer is temperature dependent, implying the protein environment is better organized for H-tunneling in the former. Our QM/MM calculations revealed that the hydride transfer has a transition state (TS) that is invariable with temperature while the proton transfer has multiple subsets of TS structures, which corroborates with our experimental results. The calculations also suggest that collective protein motions rearrange the network of H-bonds to accompany structural changes in the ligands during and between chemical transformations. These computational results not only illustrate functionalities of specific protein residues that reconcile many previous experimental observations, but also provide guidance for future experiments to verify the proposed mechanisms. In addition, we conducted experiments to examine the importance of long-range interactions in TSase-catalyzed reaction, using both kinetic and structural analysis. Those experiments found that a remote mutation affects the hydride transfer by disrupting concerted protein motions, and Mg2+ binds to the surface of TSase and affects the hydride transfer at the interior active site. Both our experiments and computations have exposed interesting features of ecTSase that can potentially provide new targets for antibiotic drugs targeting DNA biosynthesis. The relationship between protein structure, motions, and catalytic activity learned from this project may have general implications to the question of how enzymes work.