[This corrects the article DOI: 10.3389/fpls.2021.796181.].

BACKGROUND:Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.RESULTS:A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.CONCLUSIONS:The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.

The Turos laboratory has been working with N-thiolated β-lactams for years trying to understand the mode of action and structural features it needs to have biological activity. Over the years new data has shown promising inhibitory activity against various microbes. In this dissertation, a review of the vast amount of work carried out on N-thiolated β-lactams in Turos laboratory has been done and their novelty, in terms of structure and mechanism has been discussed. A complete outline of our work in the discovery and ongoing development of these compounds, starting from our initial, unexpected finding of antimicrobial activity for one of the lead compounds, to a more complete understanding of their chemical and biological mode of action and potential utility as antibacterial compounds, has been provided. Previous researches by graduate students in the Turos laboratory have shown that N-thiolated β-lactams targets Type II Fatty Acid Synthesis (FAS). In process of understanding this further, other FAS inhibiting antibiotics like Triclosan were compared to our lactams by adding excess of exogenous fatty acids. Results revealed vast differences in the MIC value of triclosan and N-thiolated β-lactams, giving an idea that there might be a different mode of action or a different target altogether. The third chapter discusses the study of attaching hydrophilic C3 side chains like amino acids and carbohydrates on N-thiolated β-lactams while studying the influence of microbiological activity. From the study it was found that the lengthening of the side chain halts the inhibitory activity regardless of whether the side chain contains unsaturation or branching. Results showed that polar groups were not well tolerated and the inhibitory activity goes down regardless of polarity. Finally, research on dual-action antibiotics was discussed. Antibiotics cause continuous bacterial resistance and in this aspect use of two drugs with different mode of action can call for reduction of the resistance. Herein, N-acyl ciprofloxacin and N-thiolated β-lactams were connected together via an ester linkage. Six new hybrid compounds have been synthesized successfully and tested against E. faecium, K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae.

Groat oil content and composition are important quality traits in oats (Avena sativa L). These traits are controlled by many genes with additive effects. The chromosomal regions containing these genes, known as quantitative trait loci (QTL), can be discovered through their close association with markers. This study investigated total oil content and fatty acid components in an oat breeding population derived from a cross between high oil ('Dal') and low oil ('Exeter') parents. A genetic map consisting of 475 DArT (Diversity Array Technology) markers spanning 1271.8 cM across 40 linkage groups was constructed. QTL analysis for groat oil content and composition was conducted using grain samples grown at Aberdeen, ID in 1997. QTL analysis for multiple agronomic traits was also conducted using data collected from hill plots and field plots in Ottawa, ON in 2010. QTLs for oil content, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) were identified. Two of the QTLs associated with oil content were also associated with all of the fatty acids examined in this study, and most oil-related QTL showed similar patterns of effect on the fatty acid profile. These results suggest the presence of pleiotropic effects on oil-related traits through influences at specific nodes of the oil synthesis pathway. In addition, 12 QTL-associated markers (likely representing nine unique regions) were associated with plant height, heading date, lodging, and protein content. The results of this study will provide information for molecular breeding as well as insight into the genetic mechanisms controlling oil biosynthesis in oat.

Groat oil content and composition are important quality traits in oats (Avena sativa L). These traits are controlled by many genes with additive effects. The chromosomal regions containing these genes, known as quantitative trait loci (QTL), can be discovered through their close association with markers. This study investigated total oil content and fatty acid components in an oat breeding population derived from a cross between high oil ('Dal') and low oil ('Exeter') parents. A genetic map consisting of 475 DArT (Diversity Array Technology) markers spanning 1271.8 cM across 40 linkage groups was constructed. QTL analysis for groat oil content and composition was conducted using grain samples grown at Aberdeen, ID in 1997. QTL analysis for multiple agronomic traits was also conducted using data collected from hill plots and field plots in Ottawa, ON in 2010. QTLs for oil content, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) were identified. Two of the QTLs associated with oil content were also associated with all of the fatty acids examined in this study, and most oil-related QTL showed similar patterns of effect on the fatty acid profile. These results suggest the presence of pleiotropic effects on oil-related traits through influences at specific nodes of the oil synthesis pathway. In addition, 12 QTL-associated markers (likely representing nine unique regions) were associated with plant height, heading date, lodging, and protein content. The results of this study will provide information for molecular breeding as well as insight into the genetic mechanisms controlling oil biosynthesis in oat.

The disease malaria afflicts more than a billion people and kills almost one to three million of them every year. Of the four species of Plasmodium affecting man viz., P. falciparum, P. vivax, P. ovale and P. malariae, Plasmodium falciparum is the deadliest as it causes cerebral malaria. The situation has become worse with the continuous emergence of drug resistance in the parasite. Therefore, improving existing drugs and deciphering new pathways for drug development are the need of the hour. The discovery of the type II fatty acid biosynthesis pathway in Plasmodium falciparum (Surolia and Surolia, 2001) has opened up new avenues for the development of new antimalarials as this pathway is entirely different from the human host in which type I pathway exists. Although many biochemical pathways such as the purine, pyrimidine and carbohydrate metabolic pathways, and the phospholipid, folate and heme biosynthetic pathways operate in the malaria parasite and are being investigated for their amenability as antimalarial therapeutic targets, no antimalarial of commercial use based on the direct use of these biochemical pathways as targets has emerged so far. This is due to the fact that the structure and function of the targets of these drugs overlaps with that of the human host. A description of such pathways forms the Chapter 1 of the thesis. This is followed by a description of the discovery and the importance of fatty acid biosynthesis pathway and the available literature on the various enzymes that are targets of potential antimalarials. Three isoforms are known for condensing enzymes - FabH which functions in initiation, and FabB and FabF which function in elongation. These isoforms differ in their biochemical properties and have unique roles to play in deciding the membrane composition of any organism. This aspect is also discussed in this chapter. Cloning and expression of -ketoacyl-ACP synthase, FabB/F from Plasmodium falciparum is described in Chapter 2. PfFabB/F is coded by the nuclear genome and is targeted to the apicoplast. The gene is coded by the locus MAL6P1.165 and the putative amino acid sequence of the protein exists in PlasmoDB. All apicoplast targeted proteins have a characteristic bipartite leader sequence consisting of a signal and a transit peptide sequence (Waller et al., 1998). Since the mature protein start site was not known and none of the software packages could predict the site, I aligned the PfFabB/F sequence with the sequences of other -ketoacyl-ACP synthases. On the basis of similarity with E. coli synthases and the mature protein start site of plant synthases, I cloned the first construct of PfFabB/F. The sequence was amplified by PCR and ligated in pET as well as pGEX vector. Expression in various hosts under different temperature and induction conditions could not solubilize the protein in significant quantities and most of the protein was found in inclusion bodies. Next I expressed the sequence with five more amino acids towards the N-terminal and expressed it as an N- terminal NusA fusion. The protein was purified by single step Ni-NTA affinity chromatography. Along with the full length protein (108 kDa), a truncated version of the protein was also obtained. The identity of the protein was confirmed by western blotting using anti-His antibody and anti-FabB/F antibody. In Chapter 3, the substrate specificity of PfFabB/F has been elucidated. PfFabB/F condenses malonyl-ACP with a range of acyl-ACPs. In vivo, acyl carrier protein (ACP) shuttles the acyl substrates between various enzymes of the fatty acid biosynthesis pathway. Enzymes of the pathway other than synthases can accept substrate analogs like acyl-CoA and acyl-NAC’s also in vitro. Acyl-ACPs are not very stable species and thus are not commercially available. Therefore, they have to be synthesized. Since malonyl-ACP could not be synthesized by chemical means, enzymatic synthesis of acyl-ACPs was done. Acyl-ACP synthetase (Aas) or holo-ACP synthase (ACPS) can be used for enzymatic synthesis. Aas is specific only for longer chain substrates; therefore, I decided to use holo-ACP synthase, an enzyme responsible for converting apo-ACP to holo-ACP in the presence of CoA in vivo (Lambalot and Walsch, 1995). When acyl-CoAs are supplied in place of CoA, acyl-ACP is produced. Malonyl-ACP and acyl-ACPs (C4-C16:1) were thus synthesized using holo-ACP synthase from E. coli. The reaction went to almost 95% completion, indicating broad substrate specificity of this enzyme. Bacterial or plant acyl-ACPs of different chain lengths can be resolved by Conformation Sensitive PAGE (Heath and Rock, 1995, Post- Beittenmiller et al., 1991). However, Pfacyl-ACPs synthesized using ACPS did not show any significant shift on CS-PAGE. Therefore I resorted to MALDI-TOF (Matrix Assisted Laser Desorption and Ionization- Time Of Flight) for monitoring the PfFabB/F condensation reactions. PfFabB/F condensed C4-C12-ACPs with malonyl-ACP to their corresponding -ketoacyl-ACP products, with C6, C8 and C10-ACPs being most readily elongated. C14-ACP was very sluggishly elongated, and C16 and C16:1-ACPs were not elongated at all. The condensation reaction was also followed by autoradiography using14C labeled malonyl-ACP, exploiting the clear mobility shift between malonyl-ACP and the other acyl-ACPs. The inhibitory effect of cerulenin, a known inhibitor of condensing enzymes was also checked. PfFabB/F also exhibited malonyl decarboxylase activity resulting in the production of acetyl-ACP in the absence of any significant condensation activity. All the enzymes of fatty acid synthesis pathway required to complete a cycle were assembled together for the in vitro reconstitution of Plasmodium fatty acid synthesis cycle which is described in Chapter 4. Earlier studies of Surolia & Surolia have shown that C12 and C14 fatty acids are the major constituents of Plasmodium lipids. One of my objectives was to determine the maximum length of the acyl ACP product that is synthesized when all the functionally active enzymes of fatty acid synthesis are put together (Kapoor et. al, 2001, Sharma et al., 2003, Karmodiya and Surolia, 2006). Condensing enzymes have a deterministic role in the fatty acid composition as they catalyze the only irreversible step in fatty acid biosynthesis. By analyzing products of the elongation cycle by mass spectrometry it was apparent that C14-ACP is the longest species formed. As already mentioned, PfFabB/F readily elongates C12-ACP but C14-ACP is weakly elongated. Thus the end product of the Plasmodium FAB pathway is influenced by the substrate specificity of PfFabB/F. This confirms the role of PfFabB/F as a decisive enzyme in determining the length of fatty acids synthesized. The inhibition of the cycle by cerulenin and triclosan is also described in this chapter. Chapter 5 describes the ability of the PffabB/F gene to complement for the mutation of condensing enzymes in CY244 cells (fabBtsfabF-, Yasuno et al., 2004). CY244 cells were transformed with pBAD alone or PfFabB/F cloned in pBAD vector (pBADPffabB/F) and the growth was monitored at non-permissive temperature. The product of PfFabB/F could rescue the growth of mutant cells at high temperature but only in the presence of oleic acid. FabB and FabF are the isoforms of condensing enzymes involved in elongation of the fatty acid synthesis cycle but they have a unique role to play (Garwin et al., 1980). FabB is responsible for unsaturated fatty acid synthesis, and fabB-mutants require oleic acid supplementation for growth. FabF is utilized in temperature regulation of membrane fluidity and E. coli FabF elevates the level of C18:1 or cis-vaccenic acid at lower growth temperature but FabF-mutants have no growth phenotype (Ulrich et al., 1983). Rescue of CY244 cells in the presence of oleic acid supplementation indicated that the PffabB/F gene behaves like FabF and not FabB. Analysis of the fatty acid composition of membrane lipids of CY244 cells transformed with pBAD vector or pBADPffabB/F by GC-MS demonstrated no elevated levels of cis-vaccenic acid in transformed cells. This observation is in agreement with the in vitro determined substrate specificity data which shows that PfFabB/F does not elongate C16:1ACP. The thesis ends with a summary of the findings in Chapter 6 in the context of FabB and FabF enzymes known from other sources. 2, 4, 4’-Trichloro-2’hydroxydiphenylether, commonly known as triclosan, has been used as a topical antibacterial agent for decades. I determined its efficacy in treating acute systemic bacterial infection in mouse model. Triclosan, as compared to other well known antibiotics, could extend the survival time of mice by 48 hours. This work is described in Appendix I. (Sharma et al., 2003)

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.

Groat oil content and composition are important quality traits in oats (Avena sativa L). These traits are controlled by many genes with additive effects. The chromosomal regions containing these genes, known as quantitative trait loci (QTL), can be discovered through their close association with markers. This study investigated total oil content and fatty acid components in an oat breeding population derived from a cross between high oil ('Dal') and low oil ('Exeter') parents. A genetic map consisting of 475 DArT (Diversity Array Technology) markers spanning 1271.8 cM across 40 linkage groups was constructed. QTL analysis for groat oil content and composition was conducted using grain samples grown at Aberdeen, ID in 1997. QTL analysis for multiple agronomic traits was also conducted using data collected from hill plots and field plots in Ottawa, ON in 2010. QTLs for oil content, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) were identified. Two of the QTLs associated with oil content were also associated with all of the fatty acids examined in this study, and most oil-related QTL showed similar patterns of effect on the fatty acid profile. These results suggest the presence of pleiotropic effects on oil-related traits through influences at specific nodes of the oil synthesis pathway. In addition, 12 QTL-associated markers (likely representing nine unique regions) were associated with plant height, heading date, lodging, and protein content. The results of this study will provide information for molecular breeding as well as insight into the genetic mechanisms controlling oil biosynthesis in oat.

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)

Malaria, caused by Plasmodium, is one of the most devastating infectious diseases of the world in terms of mortality as well as morbidity (WHO, 2002). The development of resistance in the Plasmodium falciparum against the present antimalarials has made the situation very alarming (Trape et al., 2000). To combat this situation, new antimalarials as well as identification of new drug targets are urgently required. The discovery of the presence of type II fatty acid biosynthesis system in the malarial parasite has offered several promising new targets for this mission. This thesis describes the successful cloning of fabZ from Plasmodium falciparum, its expression in E. coli, single step affinity purification, kinetic characterization and most importantly discovery of two small molecule inhibitors (Sharma et al., 2003). The study was executed to gain insights into the structure and function of PfFabZ to get better understanding of the interactions with its substrate analogs, novel inhibitors and also acyl carrier protein (PfACP). The molecular details of the interactions of the two novel inhibitors were also determined. Lastly, the residues of PfFabZ important for the interaction with PfACP were successfully elucidated. Chapter 1 presents a brief review of the literature about the disease as well as the life cycle, biology and the metabolic pathways operational in malarial parasite, Plasmodium falciaparum. The discovery of type II FAS in P. falciparum and the aims and the scope of the thesis are also discussed. The quest of developing new antimalarials, study of the mechanism of actions of antimalarials such as quinine and its derivatives along with the major metabolic pathways (Purine, pyrimidine, phospholipids, carbohydrate metabolism, folate and heme biosynthesis pathways etc.) existing in P. falciparum are described in detail in this chapter. Origin and importance of apicoplast in P. falciaprum is also described in brief. For long, it was believed that Plasmodium spp. are incapable of de novo fatty acid synthesis but this view has undergone substantial revision due to the recent discovery of plant and bacterial type of fatty acid biosynthesis pathway in them (Surolia and Surolia, 2001). As this pathway is distinct from that of the human host it has accelerated the momentum for the discovery of new antimalarials (Surolia and Surolia, 2001). The Chapter also surveys the details of type II FAS in bacteria, particularly that of E. coli (Rock and Cronan, 1996). The dehydratase step which is the third step of fatty acid elongation cycle has been covered in considerable detail. Lastly, it focuses on the recent advancement in the understanding of fatty acid biosynthesis system in Plasmodium falciparum along with some inhibitors targeting the malarial FAS. As each enzyme of the Plasmodium FAS can serve as good antimalarial targets, my work focuses on the dehydratase step catalyzed by β-hydroxyacyl-ACP dehydratase (PfFabZ). Cloning, expression and kinetic characterization of PfFabZ forms the major content of Chapter 2. The PlasmoDB data base was searched for this gene and the mined out open reading frame contained sequence of the putative FabZ together with the bipartite leader polypeptide. Our aim was to clone the mature PfFabZ without the bipartite leader sequence. Amplification of the mature pffabZ using Plasmodium falciparum genomic DNA revealed the presence of an intron in the ORF and the gene was finally cloned by RT-PCR in pET-28a(+) vector. It was expressed with an N-terminal hexahistidine tag in BL-21(DE3) cells and purified to near homogeneity but the protein was insoluble and unstable. Truncation of 12 residues from the N-terminal end improved the stability and solubility of the protein by 3-5 fold. Truncated PfFabZ was used for all future experiments. FabZs from other sources are reported to be hexamer in solution but PfFabZ showed homodimeric arrangement in the conditions used for gel filtration as well as dynamic light scattering studies. Kinetics of PfFabZ was characterized using substrate analogs, β-hydroxybutryl-CoA (forward substrate) and Crotonoyl-CoA (reverse substrate). Both the forward and reverse reaction were thoroughly characterized by spectrophotometry and HPLC and the reverse reaction was found to be 7 times faster than the forward reaction. Km οf crotonoyl-CoA was calculated to be 86 µM and kcat/Km of 220 M-1s-1 whereas the Kmfor β-hydroxybutryl-CoA was found to be 199 µM and kcat/Kmof 80.2 M-1s-1. The kinetic data clearly indicates the higher affinity of PfFabZ for the reverse substrate. Chapter 3 describes the discovery of two small molecules inhibitors, NAS-21 and NAS-91 for PfFabZ, their detailed inhibition kinetics and their effect on the growth of Plasmodium falciparum in culture. These inhibitors were the first inhibitors to be reported for FabZ class of enzymes with an IC50 ranging below 15 µM. Both of them inhibited PfFabZ following competitive kinetics with respect to the substrates utilized for both the forward and reverse reactions. The inhibition data were analyzed by Lineweaver-Burk and Dixon plots and both inhibitors showed competitive inhibition kinetics with dissociation constant in submicromolar range. Binding constants for both the inhibitors were also determined by fluorescence titration method and were calculated to be 1.6 (± 0.04) X 106 M-1 for NAS-91 and 1.2 (± 0.03) X 106 M-1 for NAS-21. These inhibitors were checked on Plasmodium falciparum culture and both inhibited parasite growth with IC50 values of 7 µM and 100 µM for NAS-21 and NAS-91, respectively. They also inhibited the incorporation of [1,2-14C]-acetate in the fatty acids of the P. falciparum conforming the inhibition of fatty acid biosynthesis. FabZ class of enzymes are thought to contain His-Glu as a catalytic dyad. Based on the disparity in the arrangement of residues at the active site of the dimeric (Swarnamukhi et al., 2006) and hexameric forms of PfFabZ in the crystal structures (Kosteriva et al., 2005), we set out to elucidate the active site residues in PfFabZ which is described in Chapter 4. The role of each of the presumed active site residues His-133 and Glu-147 along with Arg-99 and His-98 were analyzed by chemical modification studies and site directed mutagenesis. Single and double mutants were prepared and the activity of the mutants was monitored by spectrophotometry and isothermal titration calorimetry (ITC). It was concluded that in PfFabZ, His-133 and Glu-147 makes the catalytic dyad, His-98 might be important in directing the substrate in correct orientation while Arg-99 is involved in maintaining the active site loop in proper orientation rather than taking direct part in catalysis. Chapter 4 also concludes that dimeric form of PfFabZ is inactive species and turns into active hexameric form in the presence of substrate. Chapter 5 describes the molecular details of NAS-21 and NAS-91 interactions with PfFabZ. The fact that both these compounds inhibited PfFabZ in competitive manner, prompted me to examine their interaction with the residues in the active site tunnel. Apart from the His-133 and Glu-147 catalytic dyad the only polar residue is His-98 and chemical modification and site directed mutagenesis studies were done to elucidate the interactions of these residues with NAS-21 and NAS-91. Both the inhibitors were able to protect the modification of histidines by DEPC in wild type PfFabZ, His-98-Ala mutant and His-133-Ala mutant but with differential strength, indicating that they do interact with histidines. The interaction of these inhibitors was further confirmed by determining the dissociation constants of wPfFabZ, His-98-Ala, His-133-Ala, His-98-Ala/His-133-Ala double mutant, Glu-147-Ala mutant by fluorescence titration method. The results obtained from chemical modification and fluorescence titration studies confirmed that NAS-21 interacts strongly with histidines, His-98 and His-133 but not with Glu-147. On the other hand NAS-91 interacts loosely with His-98 and His-133 but strongly with Glu-147. Chapter 5 concludes with the observation that both the inhibitors (NAS-21 and NAS-91) interact with the active site residues of PfFabZ, preventing the substrate to enter the active site tunnel. Acyl carrier protein (ACP) is a small acidic protein to which the acyl chain intermediates are tethered and shuttled from one enzyme to another for the completion of fatty acid elongation cycle. Whenever acyl carrier proteins are expressed in E. coli, they are present in three forms apo, holo and acyl-ACPs. Chapter 6 describes a novel method for the expression of histidine tagged PfACP in pure holo form, protocol for the cleavage of his-tag from PfACP by thrombin preparation of homogenous singly enriched ie PfACP [15N]-labeled or [13C]-labeled PfACP as well as doubly enriched [15N]-[13C] PfACP samples for its structure elucidation by NMR (Sharma et al., 2005). These studies also constituted reporting of a holo-ACP structure from any of the sources for the first time (Sharma, et. al. 2006). The purified pure holo-PfACP was further used for the interaction studies with PfFabZ. Earlier studies have shown that ACP interacts with FAS enzymes via helix II with conserved set of residues but the molecular details of the interactions are poorly known (Zhang, et. al., 2003). We have recently solved the NMR structure (Sharma, et. al., 2006) of PfACP and crystal structure of PfFabZ (Swarnamukhi, et. al., 2006). So, both the structures were docked using Cluspro server. Chapter 7 elucidates the roles of important residues on PfFabZ surface near the active site entry which are responsible for interacting with PfACP. The residues lining the active site entry were identified and mutated. The residues lining the active site tunnel of PfFabZ are Arg102, Lys104, Lys105, Lys123, Leu94, Phe95, Ala96, Gly97, Ile128, Ile145, Phe150 and Ala151. Charged residues were mutated to alanine and also to oppositely charged residues while the neutral residues were changed to charged residues. The interaction of PfFabZ mutants with PfACP was studied by ACP independent enzymatic assay and surface plasmon resonance (SPR) spectroscopy. It was concluded that PfFabZ and PfACP interaction is mainly governed by electrostatic interaction made by the charged residues (Lys104 being the most important residue) and is fine tuned by hydrophobic interactions. Chapter 8 summarizes the findings of the thesis. FabZ from Plasmodium falciparum was cloned and biochemically characterized. Two inhibitors for this enzyme were discovered and their molecular details of binding to PfFabZ were elucidated. The presence of catalytic dyad was confirmed and finally the residues of PfFabZ important for interaction with PfACP were elucidated. Appendix I describes the inhibition of PfENR (enoyl ACP reductase), the rate limiting and the fourth enzyme of the fatty acid elongation pathway by green tea extracts. Three tea catechins (EGCG, EGC and ECG) and two plant polyphenols (quercetin and buteine) were selected for the inhibition study. All the catechins inhibited PfENR potently with Ki values in nanomolar range. Among the five compounds studied, EGCG was found to be the best inhibitor. All of them blocked the NADH binding site showing competitive kinetics with respect to NADH and uncompetitive kinetics with crotonoyl-CoA, the substrate analog. Most importantly, the catechins potentiated the inhibition of PfENR by triclosan, a well known PfENR inhibitor. We also report that in the presence of tea catechins triclosan behaves as a slow-tight binding inhibitor of PfENR. The overall inhibition constant of triclosan in the presence of EGCG was calculated to be 2pM which is 50 times better than the earlier reported values with NAD+ (Kapoor, et. al., 2004).