The thesis deals with X-ray crystallographic analysis of two enzymes involved in the fatty acid biosynthesis pathway, known as Fatty Acid Synthase or FAS, of the malarial parasite, Plasmodium falciparum, in order to understand their functions at the atomic level and to provide structural basis for the rational design of antimalarial compounds. Targeting highly specific and well-characterized biochemical pathways to develop effective therapeutic agents has the advantage of designing new drugs or modifying the existing ones based on the details of the known features of the processes. Knowledge of the three-dimensional structures of the molecules involved in the reactions will enhance the capabilities of this procedure. The recently identified fatty acid biosynthesis pathway in Plasmodium falciparum is undoubtedly an attractive target for drug development as it differs from that in humans. In the malarial parasite, each reaction of the pathway is catalyzed by a specific enzyme whereas in humans, the synthesis is carried out by a single multidomain enzyme. Essentially each step in the FAS of P. falciparum can be a potential target to prevent the growth of the parasite as the fatty acids are essential for the formation of the cell membrane which is vital for its survival. All the reactions of this pathway have been well-characterized. Nevertheless, there is a dearth of structural information of the proteins involved in performing various functions in this pathway. When this work was initiated, crystal structures of none of these proteins were reported. The current work on the plasmodial FAS proteins has been undertaken with a view to obtain precise structural details of their reaction and inhibition mechanisms. The introductory chapter of the thesis includes a discussion on malaria, the fatty acid biosynthesis in various organisms and an overview of the structural features of the enzymes involved in the pathway that have been characterized from other organisms.The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the other computational and biophysical methods used to further characterize the enzymes under study. FabI, the enoyl acyl carrier protein reductase, that regulates the third step in FAS has been crystallized as a binary complex with its cofactor NADH and as a ternary complex with NAD+and triclosan. The crystal structures of the binary and the ternary complexes have been determined at 2.5 and 2.2 ˚A, respectively. The mode of binding of the cofactor and the inhibitor triclosan to the enzyme with details of the interactions between them could be clearly obtained from these structures. Each subunit of the tetrameric FabI has the classical Rossmann fold. We carried out a thorough analysis of this structure and compared it with the FabI structures from various sources, four microbial (Escherichia coli, Mycobacterium tuberculosis and Helicobacter pylori) and one plant (Brassica napus), and arrived at a number of significant conclusions: Though the tertiary and the quaternary structures of the enzymes from different sources are similar, the substrate binding loop shows significant changes. The position and nature of the loop are strongly correlated to the affinity of triclosan to the enzyme. Small to major changes in the structure of the enzyme occur to enhance ligand binding. Water molecules play an important role in enzyme-ligand interactions. The crystal structure has also confirmed our previous prediction based on modeling studies of the enzyme that the introduction of bulkier groups at carbon 4’ of triclosan is likely to improve its efficacy as an inhibitor of FabI of P. falciparum. It has also provided the structural basis for its preference to bind to the coenzyme NADH but not to NADPH which was also predicted by our modeling studies. Chapters 3 and 4 discuss the structure solution and a comparative analysis of the crystal structures of FabIs from various sources. The crystal structure of FabZ, the β-hydroxyacyl acyl carrier protein dehydratase of P. falciparum, has been determined at a resolution of 2.4 ˚A. Each subunit of FabZ has a hotdog fold with one long central α-helix surrounded by a six-stranded antiparallel β-sheet. FabZ has been found to exist as a homodimer in the crystals of the present study in contrast to the hexameric form which is a trimer of dimers crystallized in a different condition, reported while we completed the structure of the dimeric form. In the dimeric form, the architecture of the catalytic site has been drastically altered with two catalytic histidine residues moving away from the catalytic site caused by two cis to trans peptide flips compared to the hexameric form. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate-binding. The dimeric state and an altered catalytic site architecture make the dimeric FabZ presented in the thesis distinctly different from the FabZ structures described so far. This is the first observation and report of the existence of an inactive form of the enzyme and its unique structural features. Further analysis using dynamic light scattering and size exclusion chromatographic studies have shown that a pH-related conformational switching occurs between the inactive dimers and active hexamers of FabZ in P. falciparum. These findings open alternate and entirely new strategies to design inhibitors to make FabZ inactive. FabZ crystals show polymorphism with varying length of its longest cell axis. We could collect X-ray diffraction data for three of these forms. An analysis of these forms revealed that three flexible loops of the structure including those containing the peptide flips compete for the space between two symmetry-related molecules. In the form with the longest cell axis, the loops have the highest stability resulting in a better diffraction from the crystal. We also performed docking studies with two previously characterized inhibitors of FabZ. The docking showed that the inhibitors bind strongly at the active site each one making a number of different interactions with the catalytic residues. Observations from our docking studies are in excellent agreement with and strongly supported by chemical modification and fluorimetric analysis of the wild type enzyme and its mutants. Chapters 5 and 6 explain in detail about the structure solution of dimeric form of PfFabZ, the pH induced conformational flipping of two cis-trans peptide flips that lead to different oligomeric states, and the structural basis for these oligomeric shifts. The mechanism of action of PfFabZ inhibitors NAS-21 and NAS-91 are also discussed in detail. Intrigued by the hot dog fold of the Fab enzyme, we have analyzed and compared proteins having this fold in their structures. It has been observed that the fold is often associated with fatty acids. However, the sequences, the quaternary structures, substrate specificities and the reactions that the proteins catalyze are quite diverse. The consensus sequence motifs lining the interface of quaternary association and at active site clearly indicated that the information for different modes of quaternary associations is embedded in the sequences itself. The diversity in function and quaternary association of hot dog fold proteins and their structure-function relationships are discussed in chapter 7. Malaria affects hundreds of millions of people worldwide causing about two million deaths every year. In spite of the commendable success of the available antimalarials, it has not been possible to contain the disease completely as the protozoan has become resistant to a majority of frontline drugs. The structural studies presented here should enhance the current biochemical knowledge to develop selective and more potent inhibitors of the pathway and contribute to the ongoing efforts to fight the disease.

Mit dem Beginn der Industrialisierung stieg in den westlichen Kulturen rasant die Prävalenz von Krankheitsbildern wie Adipositas, arterieller Hypertonie, Typ-2-Diabetes Mellitus und Hyperlipidämie, die als Cluster eines multifaktoriellen Krankheitsbildes namens „Metabolisches Syndrom“ (MTS) verstanden werden. Tierstudien, in denen durch die Inhibition der Fettsäuresynthase (FASN) ein rapider Abfall des Körpergewichts in Mäusen erzeilt wurden, bestätigen zunehmend genetische Prädispositionen als ursächlich für die Ausbildung des MTS. Um herauszufinden ob und in welchem Ausmaß das FASN-Gen mit humanen Merkmalen des MTS assoziiert ist, wurde das Gen in 48 nicht verwandten ostdeutschen Probanden sequenziert. Acht repräsentative tagging-SNPs wurden dabei identifiziert, anschließend in 1311 deutschen Probanden (Erwachsene) genotypisiert und in Fall-Kontroll-Studien zwischen 389 schlanken Probanden (BMI ≤ 25kg/m²) vs. 446 adipösen Teilnehmern (BMI ≥ 30kg/m²) sowie zwischen 502 glukosetoleranten Probanden (NGT) vs. 640 Probanden mit Typ-2-Diabetes (T2D) miteinader verglichen. Für den Polymorphismus rs2229422 (P = 1.3x10-5 adjustiert auf Alter, Geschlecht und Diabetes-status) konnten die stärksten Assoziationen mit BMI und weiteren Merkmalen der Fettleibigkeit identifiziert werden (adjustiert P < 0.05). Des Weiteren wurde der zuvor in der Literatur beschriebene protektive Einfluss der Val1483Ile Substitution (rs2228305) gegenüber Adipositas, sowie der geschlechts-spezifische Effekt auf den BMI bestätigt (adjustiert, P = 0.03).:1. Einleitung 6-18 1.1 Das Metabolische Syndrom (MTS) 6 1.1.1 Übergewicht im Zeitalter der Industrialisierung 6 1.1.2 Definitionen des Metabolischen Syndroms 6 1.1.3 Epidemiologie des Metabolischen Syndroms 7 1.1.4 Komponenten des Metabolischen Syndroms, deren pathophysiologische Zusammenhänge und assoziative Erkrankungen 8 1.1.5 Genese des Metabolischen Syndroms 9 1.1.5.1 Polygene Erkrankungen 10 1.1.5.2 Familien- und Zwillingsstudien 10 1.1.6 Methoden zur Identifizierung und Charakterisierung prädisponierender Gene für das MTS 11 1.1.6.1 Tiermodelle 11 1.1.6.2 Genomweite Suche 11 1.1.6.3 Kandidatengenanalyse 13 1.2 Fettsäure-Synthase/Fatty Acid Synthase (FASN) 14 1.2.1 Struktur und metabolische Funktion der Fettsäure-Synthase 14 1.2.2 Aufbau des Fettsäure-Synthase-Gen/ (FASN) und FASN-Expression 16 1.2.3 Polymorphismen des FASN-Gens und Adipositas 18 2. Zielsetzung der vorliegenden Arbeit 19 3. Probanden, Methoden und Material 20-38 3.1 Probandenkollektiv für die Genotypisierung 20 3.2 Methoden 21 3.2.1 Bestimmung der klinischen Parameter 21 3.2.2 DNA-Isolierung 23 3.2.3 Polymerase-Kettenreaktion/Polymerase Chain Reaction 23 3.2.4 Agarose-Gelelektrophorese 26 3.2.5 Polyethylenglykol-(PEG)-Fällung 27 3.2.6 DNA-Sequenzierung 28 3.2.7 Natrium-Acetat-(NaAc)-Fällung 29 3.2.8 Genotypisierung 29 3.2.9 PCR-Optimierung 31 3.2.10 Restriktions-Fragment-Längen-Polymorphismus (RFLP) 31 3.2.11 Linkage Disequilibrium (L.D.)- Analyse 33 3.2.12 Statistische Analyse 34 3.3 Material 36 4. Ergebnissteil 39-60 4.1 Ergebnisse der DNA-Sequenzierung und Identifikation der SNPs 39 4.1.1 Schematische Darstellung der Lokalisation der 16 SNPs im FASN-Gen 39 4.2 Ergebnisse der L.D.-Analyse 42 4.3 Ergebnisse der Haplotypenidentifikation 43 4.4 Ergebnisse der Assoziationsstudien 44 4.4.1 Assoziationsstudien mit Adipositas 44 Assoziationsanalyse der genetischen Varianten im FASN-Gen mit Adipositas 44 Assoziationsanalyse der Haplotypen des FASN-Gens mit Adipositas 46 Assoziationsanalyse des Polymorphismus Val1483Ile (rs2228305) mit Adipositas 47 4.4.2 Assoziationsstudien mit Typ 2 Diabetes 48 Assoziationsanalysen der genetischen Varianten im FASN-Gen mit T2D 48 Assoziationsanalysen der Haplotypen des FASN-Gens mit T2D 49 4.4.3 Assoziationsstudien mit quantitativen Merkmale des MTS 50 Assoziationsanalysen der genetischen Varianten des FASN-Gens mit quantitativen Markmalen des MTS 50 Assoziationsanalysen der Haplotypen des FASN-Gens mit quantitativen Merkmalen des MTS 52 Assoziationsanalysen des Val1483Ile Polymorphismus (rs2228305) im FASN-Gen mit quantitativen Merkmalen des MTS in den Geschlechtergruppen 52 5. Diskussion 61-66 5.1 Die Variante rs2229422 im FASN-Gen zeigt die stärksten Effekte auf Adipositas 61 5.2 Das Minor Allel A des SNPs rs17848939 schützt vor Adipositas 62 5.3 Der protektive Einfluss der Val1483Ile-Substitution bestätigt sich 63 6. Abkürzungsverzeichnis 67-69 7. Tabellenverzeichnis 70-71 8. Abbildungsverzeichnis 72 9. Zusammenfassung der Arbeit 73-76 10. Literaturverzeichnis 77-92 11. Danksagung 93 12. Eigenständigkeitserklärung 94 13. Lebenslauf 95-96

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Many cancers, including breast cancer, often develop resistance to chemotherapeutic drugs over a course of treatment. Many factors, including ABC transporter-mediated drug efflux, have been shown to play a role in acquired drug resistance. Fatty acid synthase (FASN), the key enzyme of lipid synthesis pathway, was found to be over-produced in an Adiamycin resistant breast cancer cell line MCF7/AdrVp3000, compared to its parental drug sensitive MCF7 cell line. Inhibition of FASN expression increased the drug sensitivity in breast cancer cells (MCF7/AdrVp3000 and MDA-MB-468), but not in the normal breast epithelia cell line MCF10A1. Enforced overexpression of FASN in MCF7 breast cancer cells decreased its drug sensitivity. These results indicated that FASN overexpression can induce drug resistance in breast cancers. Ectopic overexpression of FASN in MCF7 cells did not affect cell membrane permeability, transporter activity, nor did it affect cell proliferation rate. However, FASN overexpression protects cancer cells from drug-induced apoptosis by decreasing caspase-8 activation. In FASN over-expressing MCF7 cells, I discovered the positive feedback relationship between FASN and activation of Akt as previously reported. However, activation of Akt did not mediate FASN-induced drug resistance. Together with the findings that FASN expression associates with poor prognosis in several types of cancers, my investigations suggest that FASN overexpression is a novel mechanism of drug resistance in breast cancer chemotherapy. Inhibitors of FASN can be used as sensitizing agents in breast cancer chemotherapy.

Modulare Polyketidsynthasen (PKS) sind Multienzymsysteme des bakteriellen Sekundärstoffwechsels. An ihnen läuft eine schrittweise Biosynthese vielfältiger Kohlenstoff-Gerüste ab, die von einfachen Carbonsäure-Einheiten ausgeht. Polyketid-Verbindungen zeigen eine große Bandbreite pharmazeutisch interessanter Aktivitäten. In dieser Arbeit wurde eine Reihe von Evolutionsstudien durchgeführt. Zunächst wurden die phylogenetischen Beziehungen zwischen modularen PKS und anderen PKS-Systemen sowie Fettsäuresynthasen untersucht, wodurch ihre zentrale Stellung innerhalb eines langen Evolutionsprozesses gezeigt werden konnte. Eine detaillierte Analyse der Phylogenien von Domänen bakterieller modularer PKS ergab, dass das Ausmaß an Genduplikationen, Genverlusten und Ereignissen horizontalen Gentransfers zwischen den verschiedenen Bakteriengruppen beträchtlich variiert. Aus der Genomsequenz des Actinobakteriums Streptomyces avermitilis wurden die Phylogenien aller Domänentypen rekonstruiert. Der Vergleich dieser Einzelphylogenien ermöglichte es, eine Reihe von homologen Rekombinationsereignissen aufzufinden. Homologe Rekombination scheint der Hauptmechanismus zu sein, auf dem die Strukturvielfalt der Polyketide in Bakterien beruht. Mit Hilfe eines „genome mining“-Ansatzes konnte im Genom des Cyanobakteriums Nostoc punctiforme eine Reihe von Biosynthese-Clustern, die zu den PKS und nichtribosomalen Peptidsynthetasen gehören, identifiziert werden. Durch chromatographische und massenspektrometrische Analysen von Zellextrakten und Kulturüberständen konnten einige der Biosynthese-Cluster bestimmten Metaboliten zugeordnet werden. Eines der Cluster wurde hinsichtlich des produzierten Metaboliten und der Regulationsstruktur eingehender charakterisiert. Die Folgerungen aus den gewonnen Ergebnissen werden im allgemeinen Zusammenhang der Evolution metabolischer Diversität ausführlich diskutiert. / Modular polyketide synthases (PKS) are multienzym systems of bacterial secondary metabolism. They perform a stepwise biosynthesis of diverse carbon skeletons from simple carboxylic acid units. Polyketide compounds possess a wide range of pharmaceutically interesting activities. In this study, a series of evolutionary analyses was performed. Initially, the phylogenetic relationships between modular PKS and other PKS systems as well as fatty acid synthases were investigated revealing their central position within a long evolutionary process. In detail reconstruction of the phylogenies of bacterial modular PKS domains demonstrated that the extent of gene duplications, gene losses and horizontal gene transfer events varies considerably between different bacterial groups. Using the genome sequence of the actinobacterium Streptomyces avermitilis the phylogenies of all domain types were reconstructed. Comparison of these phylogenies allowed for detecting numerous events of homologous recombination, which appears to be the main mechanism underlying polyketide structural diversity in bacteria. A genome mining approach revealed a number of biosynthesis clusters of the PKS and nonribosomal peptide synthetase type in the genome of the cyanobacterium Nostoc punctiforme. Cell extracts and culture supernatants were analysed by means of liquid chromatography and mass spectrometry and some of the biosynthesis clusters could be assigned to specific metabolites. One of the clusters was characterised in greater detail regarding the produced metabolite and the cluster’s regulatory structure. The implications of the results are extensively discussed within the general context of the evolution of metabolic diversity.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid synthase (FASN) is over-expressed in many cancers, and novel inhibitors that target FASN may find use in the treatment of cancers. It has been shown that orlistat, an FDA approved drug for weight loss, inhibits the thioesterase (TE) of FASN, but can be hydrolyzed by TE. To understand the mechanisms of TE action and for designing better FASN inhibitors, I examined the mechanism of orlistat hydrolysis by TE using molecular dynamics simulations. I found that the hexyl tail of orlistat undergoes a conformational transition, destabilizing a hydrogen bond that forms between orlistat and the active site histidine. A water molecule can then hydrogen bond with histidine and become activated to hydrolyze orlistat. These findings suggest that rational design of inhibitors that block hexyl tail transition may lead to a more potent TE inhibitor. To search for novel inhibitors of TE, I performed virtual DOCK screening of FDA approved drugs followed by a fluorogenic assay using recombinant TE protein and found that proton pump inhibitors (PPIs) can competitively inhibit TE. PPIs, which are used for the treatment of gastroesophageal reflux and peptic ulcers, work to decrease gastric acid production by binding irreversibly with gastric hydrogen potassium ATPase in the stomach. Recently, PPIs have been reported to reduce drug resistance in cancer cells when used in combination with chemotherapeutics, although the mechanism of resistance reduction is unknown. Further investigation showed that PPIs are able to decrease FASN activity and cancer cell proliferation in a dose-dependent manner. These findings provide new evidence that FDA approved PPIs may synergistically suppress cancer cells by inhibiting TE of FASN and suggests that the use of PPIs in combinational therapies for the treatment of many types of cancer, including pancreatic cancer, warrants further investigation.