<p></p><p>The antifouling paint Fabi 3959 is painted on the hulls of vessels to avoid fouling caused by marine organisms attached to surfaces. The paint is registered for use on pleasure boats and other vessels weighing over 200 kg which are mainly running on the Swedish west coast (www.kemi.se).</p><p>Fabi 3959 contains copper as its active component, which is highly toxic to marine organisms and thus classified as a biocide.</p><p>Fabi antifouling paint was tested under laboratory conditions on the red macro alga <em>Ceramium tenuicorne</em>, in natural brackish water taken from the Baltic Sea. The <em>Ceramium</em> growth inhibition-test was performed using cloned algae exposed to leakage water with and without sediment. The samples containing only water held concentrations in the range of 0.11% of volume-18% of volume per liter, while the samples using sediment held doses measuring between 0.11% of volume-36% of volume leakage water per liter.</p><p>The study showed a growth inhibiting effect on the <em>Ceramium</em> in both water and sediment samples down to the lowest concentration used in the test. There was a difference between the water series and the sediment series in the EC₅₀ values of the leakage water. The mean EC₅₀value was almost 10 times lower within the sediment series compared to the water series (0.114±0.10 and 1.024±0.75, respectively). This indicates that the sediment series are more toxic to <em>Ceramium</em> than the water series. However, if the mean values of EC₅₀ are expressed as copper-concentration, there is no clear difference between the two series (0.59 ± 0.13µg/l for the sediment series and 0.62 ± 0.12 µg/l for the water series). Apparently, the test did not indicate that the sediment was absorbing the copper. Instead it cannot be excluded that another substance involved could have a growth inhibiting impact on <em>Ceramium</em>.</p><p> </p> / <p> </p><p>Båtbottenfärgen Fabi 3959 målas på fartygsskrov för att undvika påväxt av marina organismer. Färgen är registrerad att användas på fritidsbåtar och andra fartyg med en egenvikt på över 200 kg och med huvudsaklig fart på Västkusten (www.kemi.se). Den aktiva komponenten i Fabi 3959 är koppar, vilket är mycket giftigt för marina organismer och därför klassificeras den som en biocid.</p><p>Fabi båtbottenfärg testades i laborativ miljö, på den röda makroalgen <em>Ceramium tenuicorne</em> i naturligt brackvatten från Östersjön. Ett tillväxthämningstest utfördes på <em>Ceramium</em>-kloner vilka exponerades för lakvatten i bägare med och utan sediment. Proverna endast innehållande vatten bestod av koncentrationer i intervallen 0,11-18 volym% per liter medan proverna med sedimentvatten hade koncentrationer på 0,11-36 volym% per liter.</p><p>Studien visade på en signifikant tillväxthämningseffekt på <em>Ceramium</em> i både vatten och sediment, ner till den lägsta använda koncentrationen. Det förelåg en tydlig skillnad mellan vattenserierna och sedimentserierna med avseende på resultaten av EC₅₀-värdena på lakvattnet; EC₅₀-värdena av den toxiska nivån för lakvattnet visade sig ligga tio gånger lägre i sedimentserierna än i vattenserierna (0,114 ± 0,10 i sediment och 1,024 ± 0,75 i vatten). Medelvärdena av EC₅₀för koppar visar inte någon påtaglig skillnad mellan serierna (0,59 ± 0,13µg/l för sediment 0,62 ± 0,12 µg/l för vatten). Testet indikerade därmed inte att sedimentet absorberade koppar. Istället föreligger en möjlighet att en annan substans orsakade tillväxthämning på<em> Ceramium.</em></p><p> </p>

The antifouling paint Fabi 3959 is painted on the hulls of vessels to avoid fouling caused by marine organisms attached to surfaces. The paint is registered for use on pleasure boats and other vessels weighing over 200 kg which are mainly running on the Swedish west coast (www.kemi.se). Fabi 3959 contains copper as its active component, which is highly toxic to marine organisms and thus classified as a biocide. Fabi antifouling paint was tested under laboratory conditions on the red macro alga Ceramium tenuicorne, in natural brackish water taken from the Baltic Sea. The Ceramium growth inhibition-test was performed using cloned algae exposed to leakage water with and without sediment. The samples containing only water held concentrations in the range of 0.11% of volume-18% of volume per liter, while the samples using sediment held doses measuring between 0.11% of volume-36% of volume leakage water per liter. The study showed a growth inhibiting effect on the Ceramium in both water and sediment samples down to the lowest concentration used in the test. There was a difference between the water series and the sediment series in the EC50 values of the leakage water. The mean EC50 value was almost 10 times lower within the sediment series compared to the water series (0.114±0.10 and 1.024±0.75, respectively). This indicates that the sediment series are more toxic to Ceramium than the water series. However, if the mean values of EC50 are expressed as copper-concentration, there is no clear difference between the two series (0.59 ± 0.13µg/l for the sediment series and 0.62 ± 0.12 µg/l for the water series). Apparently, the test did not indicate that the sediment was absorbing the copper. Instead it cannot be excluded that another substance involved could have a growth inhibiting impact on Ceramium. / Båtbottenfärgen Fabi 3959 målas på fartygsskrov för att undvika påväxt av marina organismer. Färgen är registrerad att användas på fritidsbåtar och andra fartyg med en egenvikt på över 200 kg och med huvudsaklig fart på Västkusten (www.kemi.se). Den aktiva komponenten i Fabi 3959 är koppar, vilket är mycket giftigt för marina organismer och därför klassificeras den som en biocid. Fabi båtbottenfärg testades i laborativ miljö, på den röda makroalgen Ceramium tenuicorne i naturligt brackvatten från Östersjön. Ett tillväxthämningstest utfördes på Ceramium-kloner vilka exponerades för lakvatten i bägare med och utan sediment. Proverna endast innehållande vatten bestod av koncentrationer i intervallen 0,11-18 volym% per liter medan proverna med sedimentvatten hade koncentrationer på 0,11-36 volym% per liter. Studien visade på en signifikant tillväxthämningseffekt på Ceramium i både vatten och sediment, ner till den lägsta använda koncentrationen. Det förelåg en tydlig skillnad mellan vattenserierna och sedimentserierna med avseende på resultaten av EC50-värdena på lakvattnet; EC50-värdena av den toxiska nivån för lakvattnet visade sig ligga tio gånger lägre i sedimentserierna än i vattenserierna (0,114 ± 0,10 i sediment och 1,024 ± 0,75 i vatten). Medelvärdena av EC50 för koppar visar inte någon påtaglig skillnad mellan serierna (0,59 ± 0,13µg/l för sediment 0,62 ± 0,12 µg/l för vatten). Testet indikerade därmed inte att sedimentet absorberade koppar. Istället föreligger en möjlighet att en annan substans orsakade tillväxthämning på Ceramium.

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.

The emergence of drug resistant strains of Plasmodium has given a new face to the old disease, malaria. One of the approaches is to block metabolic pathways of the pathogen. The current thesis describes the X-ray crystallographic analysis of two enzymes of the fatty acid biosynthesis pathway of the malaria parasite Plasmodium falciparum. In order to understand the functional mechanism and mode of inhibitor binding, enzyme-inhibitor complexes were characterized, which could help in further improvement of the efficacy of the inhibitors and hence to fight against the disease. The introductory chapter of the thesis presents a discussion on malaria and different metabolic pathways of the pathogen which could be suitable targets for novel antimalarials. In continuation to that, the pathway of our choice the fatty acid biosynthesis and an overview of the structural features of the enzymes involved in the pathway that have been characterized from different organisms are also described. The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the biochemical, biophysical and other computational methods used to further characterize the enzymes under study. Triclosan, a well known inhibitor of Enoyl Acyl Carrier Protein Reductase (FabI) from several pathogenic organisms, is a promising lead compound to design effective drugs. The X-ray crystal structures of Plasmodium falciparum FabI (PfFabI), in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations, were determined and compared with triclosan binding which form the basis of chapter 3. The structures revealed that 4 and 2’ substituted compounds have more interactions with the protein, cofactor and solvent molecules as compared to triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2’ position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water based inhibitor design. 2’ and 4’ unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensates for the lost interactions due to the unsubstitution at 2’ and 4’. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2’ and 4’ positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors. Certain residues in the substrate binding tunnel of PfFabI were mutated to identify the role of these residues in substrate binding and protein stability, which forms the 4th chapter of the thesis. The substrate binding site residue Ala372 of PfFabI has been mutated to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli FabI (EcFabI) which has a Methionine at the structurally similar position of Ala372 of PfFabI. Kinetic studies of the mutants of PfFabI and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a 3-fold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. A second set of mutants generated to check this hypothesis exhibited loss of function, except in one case where, the crystal structure showed that the substrate binding loop is stabilized by a water bridge network. The main focus of chapter 5 is β-Hydroxyacyl-acyl carrier protein dehydratase of Plasmoduim falciparum (PfFabZ) which catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ was available in its hexameric (active) and dimeric (inactive) forms. However, until now PfFabZ has not been crystallized with any bound inhibitors. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of three of these complexes. This is the first report of the crystal structures of PfFabZ with competitive inhibitor complexes and the first such study on any FabZ enzyme with active site inhibitors. These inhibitors in the active site stabilize the substrate binding loop, revealing the substrate binding tunnel with an overall shape of “U”. In the crystal structure, the residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed, implying that it controls the length of the tunnel and makes it suitable for accommodating longer substrates merely by changing its side chain conformation. The hydrophobic nature of the substrate binding channel signifies the specificity for the hydrophobic tail of fatty acid substrates. The volume of the active site tunnel is determined by the sequence as well as by the conformation of the substrate binding site loop region and varies between organisms for accommodating fatty acids of different chain lengths. All PfFabZ inhibitors reported here bind to the active site through specific contacts like hydrogen bonds with catalytic residues and hydrophobic interactions. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme, that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms. The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. The analysis discussed in chapter 6, led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects. Rv0098, predicted to be the FabZ of Mycobacterium tuberculosis, was cloned, expressed, purified, crystallized, and X-ray diffraction data were collected. Molecular replacement trials with all “hot dog” fold proteins failed to yield any significant solution due to the low sequence similarity (<20%) of Rv0098 compared to other FabZs. During the trials of structure solution by multiple isomorphous replacement method, structure of Rv0098 was published and it was shown to be a long-chain fatty acyl-CoA thioesterase (FcoT). The crystal structure of Rv0098 did not explain the molecular basis of substrate specificity of varying chain lengths. Molecular dynamics studies were carried out, which revealed that certain residues of the substrate binding tunnel are flexible and thus modulates the length of the tunnel. Flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. The structural basis of accommodating long chain substrates by Rv0098 is discussed in chapter 7, by combining the crystallographic and molecular dynamics studies. Part of the work presented in the thesis has been reported in the following publications. Karmodiya, K., Sajad, S., Sinha, S., Maity, K., Suguna, K. and Surolia, N. (2007) Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase. IUBMB Life 59, 441-9. Pidugu, L. S., Maity, K., Ramaswamy, K., Surolia, N. and Suguna, K. (2009) Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins. BMC Struct Biol 9, 37. Kapoor, N., Banerjee, T., Babu, P., Maity, K., Surolia, N. and Surolia, A. (2009) Design, development, synthesis, and docking analysis of 2'-substituted triclosan analogs as inhibitors for Plasmodium falciparum enoyl-ACP reductase. IUBMB Life 61, 1083-91. Maity, K., Bhargav, S. P., Sankaran, B., Surolia, N., Surolia, A. and Suguna, K. (2010) X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibition. IUBMB Life 62, 467-76. Maity, K., Banerjee, T., Narayanappa, P., Surolia, N., Surolia, A. and Suguna, K. (2010) Effect of substrate binding loop mutations on the structure, kinetics and inhibition of Enoyl Acyl Carrier Protein Reductase from Plasmodium falciparum. (Communicated) Maity, K., Bharat, S. V., Kapoor, N., Surolia, N., Surolia, A. and Suguna, K. (2010) Insights into the functional and inhibitory mechanism of the β-Hydroxyacyl-Acyl Carrier Protein Dehydratase of Plasmodium falciparum from the crystal structures of its complexes with active site inhibitors. (Communicated)