<div>L-phenylalanine (Phe) is an important amino acid which is the precursor of various plant secondary metabolisms. Its biosynthesis and consumption are governed by different levels of regulatory mechanisms, yet our understanding to them are still far from complete. The plant has evolved a complex regulation over Phe, likely due to the fact that a significant portion of carbon assimilated by photosynthesis is diverted to its downstream products. In particular, lignin as one of them, is among the most abundant polymers in plant secondary cell wall. Studies have unraveled the interconnected metabolism involved in lignin biosynthesis, and a hierarchical gene regulatory network on top of it is also being uncovered by different research groups. These biological processes function together for sufficient lignification to ensure cell wall hydrophobicity and rigidity for plant normal growth. Yet on the other hand, the presence of lignin hinders the efficient saccharification process for biofuel production. Therefore, it is fundamental to understand lignin biosynthesis and its upstream Phe biosynthesis in a systematic way, to guide rational metabolic engineering to either reduce lignin content or manipulate its composition <i>in planta</i>.</div><div> </div><div> Phe biosynthesis was predominantly existed in plastids according to previous studies, and there exists a cytosolic synthetic route as well. Yet how two pathways are metabolically coordinated are largely under-explored. Here I describe a flux analysis using time course datasets from ¹⁵N L-tyrosine (Tyr) isotopic labeling studies to show the contributions from two alternative Phe biosynthetic routes in Petunia flower. The flux split between cytosolic and plastidial routes were sensitive to genetic perturbations to either upstream chorismate mutase within shikimate pathway, or downstream plastidial cationic amino-acid transporter. These results indicate the biological significance of having an alternative biosynthetic route to this important amino acid, so that defects of the plastidial route can be partially compensated to maintain Phe homeostasis.</div><div> </div><div> To understand the metabolic dynamics of the upstream part of lignin biosynthesis, we developed a multicompartmental kinetic model of the general phenylpropanoid metabolism in Arabidopsis basal lignifying stems. The model was parameterized by Markov Chain Monte Carlo sampling, with data from feeding plants with ring labeled [¹³C₆]-Phe. The existence of vacuole storage for both Phe and <i>p</i>-coumarate was supported by an information theoretic approach. Metabolic control analysis with the model suggested the plastidial cationic amino-acid transporter to be the step with the highest flux controlling coefficient for lignin deposition rate. This model provides a deeper understanding of the metabolic connections between Phe biosynthesis and phenylpropanoid metabolism, suggesting the transporter step to be the promising target if one aims to manipulate lignin pathway flux.</div><div> </div><div> Hundreds of gene regulatory interactions between transcription factors and structural genes involved in lignin biosynthesis has been reported with different experimental evidence in model plant Arabidopsis, however, a public database is missing to summarize and present all these findings. In this work, we documented all reported gene regulatory interactions in Arabidopsis lignin biosynthesis, and ended up with a gene regulatory network consisting of 438 interactions between 72 genes. A network is then constructed with linear differential equations, and its parameters were estimated and evaluated with RNA-seq datasets from 13 genetic backgrounds in Arabidopsis basal stems. We combined this network with a kinetic model of lignin biosynthesis starting from Phe and ending with all monolignols participated in lignin polymerization. This hierarchical kinetic model is the first model integrating dynamic information between transcriptional machinery and metabolic network for lignin biosynthesis. We showed that it is able to provide mechanistic explanations for most of experimental findings from different genotypes. It also provides the opportunity to systematically test all possible genetic manipulation strategies targeting to lignification relevant genes to predict the lignin phenotypes <i>in silico</i>.</div>

by Lee Chi-Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 74-78). / Abstracts in English and Chinese. / Acknowledgment --- p.ii / Table of Contents --- p.iii / Abstract --- p.v / Abstract (Chinese Version) --- p.vi / Abbreviation --- p.vii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- General Background --- p.1 / Chapter 1.1.1 --- Valienamine --- p.2 / Chapter 1.1.2 --- Valienamine Derivatives --- p.4 / Chapter 1.2 --- Mechanistic Aspects of Glycosidase Inhibition --- p.6 / Chapter 1.2.1 --- General Background --- p.6 / Chapter 1.2.2 --- Mechanism of Enzyme Catalyzed Hydrolysis of Glycosides --- p.6 / Chapter 1.2.3 --- Types of Glucosidase Inhibitors --- p.7 / Chapter 1.2.4 --- Inhibition of Glycosidases by Valienamine Derivatives --- p.8 / Chapter 1.3 --- Previous Synthesis of Valienamine --- p.9 / Chapter 1.3.1 --- Enantiospecific Synthesis of Valienamine by Vasella and co-workers --- p.9 / Chapter 1.3.2 --- Enantiospecific Synthesis of Valienamine by Tatsuta and co-workers --- p.10 / Chapter 1.3.3 --- Synthesis of N-Alkyl Derivatives of Valienamine --- p.12 / Chapter 1.4 --- Previous Syntheses of Valienamine-containing Pseudodisaccharides and its Diastereomers --- p.12 / Chapter 1.4.1 --- Epoxide aminolysis --- p.12 / Chapter 1.4.2 --- Condensation of amine with ketone --- p.15 / Chapter 1.4.3 --- Synthesis of pseudoaminodisaccharide by Kapp and co-workers --- p.16 / Chapter 2. --- Results and Discussion --- p.18 / Chapter 2.1 --- General Strategy --- p.18 / Chapter 2.2 --- Syntheses of coupling precursors --- p.20 / Chapter 2.2.1 --- Syntheses of protected valienamine 65 and its 2-epimer80 --- p.20 / Chapter 2.2.1.1 --- Synthesis of Diol68 --- p.20 / Chapter 2.2.1.2 --- Synthesis of Diol67 --- p.21 / Chapter 2.2.1.3 --- Synthesis of protected valienamine 65 and its 2-epimer80 --- p.22 / Chapter 2.2.2 --- Syntheses of 6-deoxyaminosugars 63 and118 --- p.24 / Chapter 2.2.2.1 --- Synthesis of benzyl ether91 --- p.24 / Chapter 2.2.2.2 --- Synthesis of β-diol103 --- p.28 / Chapter 2.2.2.3 --- Synthesis of α-alcohol107 --- p.30 / Chapter 2.2.2.4 --- Synthesis of β-diol113 --- p.31 / Chapter 2.2.2.5 --- Syntheses of amines 63 and118 --- p.33 / Chapter 2.2.3 --- Syntheses of allylic chlorides --- p.34 / Chapter 2.3 --- Syntheses of pseudoaminodissaccharides --- p.36 / Chapter 3. --- Conclusion --- p.42 / Chapter 4. --- Experimental --- p.44 / Chapter 5. --- References --- p.74 / Chapter 6. --- Appendix --- p.79 / List of spectra --- p.79

Aspartokinase is the first enzyme of the aspartate family amino acids biosynthetic pathway. Cephamycin C is a &amp / #946 / -lactam antibiotic produced as a secondary metabolite via the enzymatic reactions in the lysine branch of this pathway in Streptomyces clavuligerus. The aspartokinase activity of S. clavuligerus is under concerted feedback inhibition by two of the end product amino acids, lysine plus threonine. It is also known that carbon flow through the lysine branch of the aspartate pathway is rate limiting step in the formation of cephamycin C. Therefore, genetic alterations in the regulatory regions of the aspartokinase enzyme are expected to lead to an increased cephamycin C production. The aim of this study was to obtain S. clavuligerus mutants that possess aspartokinase enzyme insensitive to feedback inhibition by lysine and threonine, identification of the mutation(s) accounting for the resistance being the ultimate goal. For this aim, chemical mutagenesis was employed to increase random mutation rate and a population of lysine anti-metabolite resistant S. clavuligerus mutants that can grow in the presence of S-(2-aminoethyl)-L-cysteine was obtained. The mutants were screened for their aspartokinase insensitivity via enzyme assays and one mutant exhibiting the highest level of deregulation was assessed for its cephamycin C production. The results revealed a 2-fold increase in specific production of the antibiotic. As a member of &amp / #946 / -lactam class antibiotics, cephamycin C has an importance in medicine. Therefore, the mutant strain obtained might be a candidate for industrial production of the compound.

This thesis examined the enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from the pathogen Mycobacterium tuberculosis. DHDPS is a validated antibiotic target for which no potent inhibitor based on substrates, intermediates or product has been found. The importance of the homotetrameric quaternary structure in E. coli DHDPS has been demonstrated by the 100-fold decrease in activity observed in a dimeric variant, DHDPS-L197Y, created by site-directed mutagenesis. This suggested a new approach for inhibitor design: targeting the dimer-dimer interface and disrupting tetramer formation. DHDPS catalyzes the first committed step in the biosynthetic pathway of meso-diaminopimelic acid, a critical component of the mycobacterial cell wall. In this study, wild-type M. tuberculosis DHDPS was thoroughly characterized and compared with the E. coli enzyme. A coupled assay was used to obtain the kinetic parameters for M. tuberculosis DHDPS: KM(S) ASA = 0.43 (±0.02) mM, KMpyruvate = 0.17 (±0.01) mM, and kcat = 138 (±2) s 1. Biophysical techniques showed M. tuberculosis DHDPS to exist as a tetramer in solution. This is consistent with the crystal structure deposited as PDB entry 1XXX. The crystal structure of M. tuberculosis DHDPS showed active-site architecture analogous to E. coli DHDPS and a dimeric variant of M. tuberculosis DHDPS was predicted to have reduced enzyme activity. A dimeric variant of M. tuberculosis DHDPS was engineered through a rationally designed mutation to analyze the effect of disrupting quaternary structure on enzyme function. A single point mutation resulted in a variant, DHDPS-A204R, with disrupted quaternary structure, as determined by analytical ultracentrifugation and gel-filtration chromatography. DHDPS-A204R was found to exist in a concentration-dependent monomer-dimer equilibrium, shifted towards dimer by the presence of pyruvate, the first substrate that binds to the enzyme. The secondary and tertiary structure of DHDPS-A204R was analogous to wild-type M. tuberculosis DHDPS as judged by circular dichroism spectroscopy and X ray crystallography, respectively. Surprisingly, this disrupted interface mutant had similar activity to the wild type enzyme, with a kcat of 119 (±6) s-1; although, the affinity for its substrates were decreased: KM(S) ASA = 1.1 (±0.1) mM, KMpyruvate = 0.33 (±0.03) mM. These results indicated that disruption of tetramer formation does not provide an alternative direction for drug design for DHDPS from M. tuberculosis. Comparison with the recently discovered dimeric DHDPS from Staphylococcus aureus shed further light on the role of quaternary structure in DHDPS. In M. tuberculosis DHDPS-A204R and the naturally dimeric enzyme, the association of monomers into the dimer involves a greater buried surface area and number of residues than found in E. coli DHDPS-L197Y. This provides a framework to discriminate which DHDPS enzymes are likely to be inactive as dimers and will direct future work targeting the dimer-dimer interface of DHDPS as an approach for drug design.

Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica.   A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica.  Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro.   Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase. / QC 20110531

Indian Institute Of Science / Proteins associated with metabolic pathways have received considerable attention in an effort to understand the molecular details of the complex reactions catalyzed in vivo. Enzymes belonging to these pathways have also been explored as potential targets for therapeutic intervention. The L-Lysine biosynthesis pathway in particular remains an attractive target for the design of new anti-microbial compounds. The rationale for this interest stems from the finding that the pathway for Lysine biosynthesis is only present in bacteria and is absent in humans. m-DAP/L-Lysine is an essential component of the bacterial cell wall. The L-Lysine biosynthesis pathway in bacteria is fairly diverse. Three major routes for the biosynthesis of m-DAP/L-Lysine are currently known. These are the succinylase, the acetylase and the dehydrogenase pathways. The results reported in this thesis are based on enzymes involved in the L-Lysine biosynthesis pathway of a nosocomial pathogen, Staphylococcus aureus spp. COL. The structural and biochemical characterization of three enzymes, Dihydrodipicolinate synthase (DapA), Dihydrodipicolinate reductase (DapB) and Succinyl diaminopimelate desuccinylase (DapE) provide a mechanistic rationale for the activity of these proteins. These studies reveal substantial differences in the regulatory mechanisms of these enzymes that could potentially be utilized for the design of inhibitors that specifically target this biosynthesis route. This thesis is organized as follows: Chapter 1 provides an introduction to the topic of this thesis. The first part of this chapter describes the characteristic features of Staphylococcus aureus and aspects relating to the identification, pathogenesis and genomic differences in S. aureus strains. The general features of the peptidoglycan layer of the bacterial cell wall including its structure and composition are also discussed. The second part of this chapter provides an overview of the Lysine biosynthesis pathway and details of the enzymes in this pathway. Chapter 2 describes the structural and functional features of Dihydrodipicolinate synthase (DHDPS) also referred to as DapA. DHDPS catalyzes the first committed step of L-Lysine biosynthesis. Here we report the crystal structure of the native and pyruvate complexes of S. aureus DHDPS. S. aureus DHDPS is a dimer, both in solution as well as in the crystal. The functional characterization of S. aureus DHDPS revealed that this enzyme is active as a dimer. This feature distinguishes the S. aureus enzyme from the E. coli homologue where a tetrameric quaternary arrangement is essential for the activity of this protein. A comparison between the native and pyruvate-bound structures also provides a structural basis for the ping-pong reaction mechanism of this enzyme whereby the catalytic triad is drawn closer to facilitate proton transfer upon pyruvate binding. It was also noted that unlike the E. coli homologue, S. aureus DHDPS is not feedback inhibited by lysine. The lack of feedback inhibition in S. aureus DHDPS could be attributed to a unique allosteric site. The different quaternary arrangement and a distinct allosteric pocket in this enzyme thus provide a structural template for the design of specific inhibitors for this enzyme. Chapter 3 is based on preliminary studies of Dihydrodipicolinate reductase (DHDPR), encoded by the dapB gene. DHDPR catalyzes the second committed step in m-DAP/L-Lysine biosynthesis. The dapB gene encoding DHDPR was cloned and over-expressed in E. coli. Two variations of the recombinant protein were examined- one with a hexa-histidine tag at the C-terminus and the other without any tag. The recombinant DHDPR with the C-terminal hexa-histidine tag was purified by Ni-affinity chromatography and was subsequently crystallized. However, data sets collected on these crystals could not be examined further due to pronounced pseudo-translational symmetry and poor resolution. The recombinant DHDPR protein without an expression tag was purified by anion-exchange and size exclusion chromatography. Analytical gel filtration studies with recombinant DHDPR is consistent with a tetrameric quaternary arrangement of DHDPR subunits with a calculated molecular mass of 135 kDa. Diffraction data were collected to 3.3 Å resolution on crystals of apo DHDPR. These crystals belong to the C-centered monoclinic space group (C2) with unit cell parameters a = 63.17 Å, b = 78.91 Å, c = 128.38 Å and γ = 110.0°. Assuming two molecules in the asymmetric unit, the calculated Matthew’s coefficient (Vm) was 2.32 Å3 Da-1 and solvent content was 47.0 %. Molecular replacement (MR) trials with a model combining E .coli DHDPR structures (residues 1-106 of 1ARZ and 108-241 of 1DIH) as a search model resulted in a successful MR solution with two molecules in the asymmetric unit. Chapter 4 is based on the structure and regulatory mechanism of DapE also referred to as Sapep. This enzyme belongs to the M20 family of proteases and is characterized by diverse substrate specificity and multiple functional roles. These include Succinyl diaminopimelate desuccinylase, a Mn2+-dependent di-peptidase and a -lactamase. The chemical reaction involved in all these functions is broadly similar and involves amide bond hydrolysis. In an effort to understand the structure and regulatory features of this enzyme, the structure of Sapep was determined both in a Mn2+-bound form and in a metal-free (apo) form. A comparison between these structures revealed that large inter-domain movements potentially regulate the activity of this enzyme. These structures also revealed an additional regulatory mechanism wherein the inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys155 and Cys178 are not active site residues, the reduced form of this enzyme is substantially more active as a peptidase. The characterization of disulfide bond in the apo-form of the protein in solution by mass spectrometric studies and the requirement of a reducing agent for optimal catalytic activity of this protein suggests that the conformational features noted in crystal structures are also likely in solution. The structural and biochemical features of this enzyme thus provide a basis to rationalize the multiple functional roles of this protein with potential applications to MRSA-specific therapeutic strategies. Chapter 5 provides a summary of the biochemical and structural data on the three enzymes of the L-Lysine biosynthesis pathway in S. aureus spp. COL. The emphasis of the discussion in this chapter is on features that are specific to these S. aureus enzymes. The latter part of this chapter is based on the scope of future studies in this area. The appendix sections of this thesis are based on a project involving the catalytic domain of a receptor protein tyrosine phosphatase CRYP-2/cPTPRO. Appendix-I includes the structure-function analysis of the catalytic domain of CRYP-2/cPTPRO. CRYP-2/cPTPRO is a receptor protein tyrosine phosphatase (PTP) that is selectively expressed in neurons and has been implicated in axon growth and guidance. The extracellular receptor domain of this protein has eight fibronectin typeIII repeat regions while the intracellular region consists of a catalytic PTP domain. The crystal structure of CRYP-2 revealed two molecules of the catalytic domain in the asymmetric unit. The substantial buried surface area of this crystallographic oligomer suggested a homo-dimer of the catalytic domain. Solution studies however suggested that this protein is a monomer in solution based on the elution profile of CRYP-2 in a size exclusion chromatography experiment. The monomeric nature of CRYP-2 thus suggests that dimerization induced modulation of enzyme activity, such as that seen in RPTP- where a helix-turn-helix segment of one monomer blocks the active site of the other, is not possible in the case of CRYP-2. Both monomers of CRYP-2 reveal a nitrate ion bound at the active site. An advantage provided by the crystallographic dimer of CRYP-2 was that it allowed us to visualize this protein with its active site lid (WPD-loop) in both the open and closed conformations. A structural comparison of CRYP-2 with other PTP’s suggests that minor conformational rearrangements, as opposed to dimerization, could serve to regulate the activity of members of the type III family of RPTPs. Appendix-II describes a project to examine the feasibility of utilizing mesoporous matrices of alumina and silica for the controlled inhibition of enzymatic activity. In these studies, we employed bare and functionalized mesoporous alumina and MCM48 silica to deliver para-nitrocatechol sulfate (pNCS), a potent competitive PTP inhibitor. pNCS was chosen as model inhibitor because of the ease of monitoring its release using UV-Visible absorption spectroscopy. CRYP-2 was used as model enzyme in this analysis. Inhibition of catalytic activity was examined using the sustained delivery of para-nitrocatechol sulfate (pNCS) from bare and amine functionalized MCM48 and Al2O3. Among the various mesoporous matrices employed, amine functionalized MCM48 exhibited the best controlled release of pNCS and inhibition of CRYP-2. Appendix-3 incorporates additional methodologies and technical details that could not be included in the main body of the thesis.

A &#946;-D-galactofuranose é um componente de várias macromoléculas de muitos organismos, incluindo bactérias, protozoários e fungos. Interessantemente este carboidrato não usual está ausente de glicoconjugados de mamíferos. Um método alternativo foi utilizado para purificar a &#946;-D-galactofuranosidase utilizando p-nitrofenil-&#946;-D-galactofuranosideo como substrato. O meio de cultura concentrado do fungo Penicillium fellutanum foi cromatografado em coluna de DEAE-Sepharose CL 6B, seguido de cromatografia em coluna de afinidade de 4-aminofenil-1-tio-&#946;-D-galactofuranosideo-Sepharose, que permitiu a separação de dois picos com atividade enzimática após a eluição com D-galactônico-&#947;-lactona em um gradiente de 100 a 500 mM de NaCl. Os dois picos resultaram, quando analisados por SDS-PAGE, em um polipeptídeo de 70 kDa. Anticorpos produzidos contra a mistura das bandas recortadas do gel são capazes de imunoprecipitar 77 % das unidades totais de enzima. Uma das bandas recortadas foi submetida a microseqüenciamento, obtendo-se seqüências para 3 peptídeos (65, 73, 101). Anticorpos foram produzidos contra os peptídeos sintéticos e usados para imunoprecipitar um polipeptídeo com atividade de &#946;-D-galactofuranosidase. Para verificar a presença de &#946;-D-galactofuranosidase em T. cruzi, usamos a mesma coluna de afinidade de 4-aminofenil-1-tio-&#946;-D-galactofuranosideo-Sepharose. Um extrato de formas epimastigotas marcadas metabolicamente com 35S-metionina foi aplicado na coluna de afinidade. A enzima foi eluída após a lavagem com D-galactônico-&#947;-lactona em um gradiente de NaCl 100 a 500 mM. A análise por SDS-PAGE do material eluido mostrou um polipeptídeo de massa molecular aparente 50-60 kDa que é reconhecido por anticorpos anti-&#946;-D-galactofuranosidase. Os anticorpos reagem por imunoflorescência indireta também com parasitas fixados com paraformaldeído. O polipeptídio de 50-60 kDa foi empregado em ensaios de atividade enzimática. Praticamente nenhuma atividade foi detectada utilizando-se p-nitrofenil-&#946;-D-galactofuranosideo como substrato. A LPPG (lipopeptideofosfoglicana), um glicoconjugado isolado de epimastigotas de T. cruzi foi empregada como substrato. A galactopiranose, produto da hidrólise da atividade enzimática, foi detectada por cromatografia de troca iônica em alto pH sugerindo a presença de &#946;-D-galactofuranosidase em T.cruzi. / &#946;-D-galactofuranose is a component of several galactofuranose containing macromolecules of many organisms, including bacteria, protozoa and fungi. Interestingly, this unusual sugar is absent in mammalian glycoconjugates. An alternative and fast method for the purification of &#946;-D-galactofuranosidase was developed using p-nitrophenyl-&#946;-D-galactofuranoside as substrate. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE-Sepharose CL 6B followed by an affinity chromatography column of 4-aminophenyl-1-thio-&#946;-D-galactofuranoside-Sepharose which yielded two separate peaks of enzyme activity when elution was performed with 10mM D-galactonic acid-&#947;-lactone in a 100-500 mM NaCl gradient. Both peaks rendered a single 70 kDa protein as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable to immunoprecipitate 77 % units out of total units of the enzyme from the crude extract. One of the excised bands was submited to microsequencing and the sequence for 3 peptides (65, 73, 101) was obtained. Antibodies were raised against the corresponding synthetic peptides and used to immunoprecipitade a polypeptide with &#946;-D-galactofuranosidase activity. To verify the presence of &#946;-D-galactofuranosidase in T. cruzi we used the same 4-aminophenyl-1-thio-&#946;-D-galactofuranoside-Sepharose affinity column. An extract of epimastigotes metabolically labelled with 35S-methionine was applied on the affinity colunm. After washing, the putative enzyme was eluted by 10 mM D-galactonic acidy-&#947;-lactone and 100-500 mM NaCl gradient. SDS-PAGE analysis of the eluted material show a polypeptide with an apparent molecular mass of 50-60 kDa that is recognized by all the antibodies raised against &#946;-D-galactofuranosidase from P. fellutanum. Also the antibodies react with p-formaldehyde fixed parasites as detected by indirect immunofluorescence. The 50-60 kDa polypeptide was then employed in enzymatic assays. Almost no enzymatic activity was observed when p-nitrophenyl galactofuranoside was employed as substrate. LPPG (lipopeptidophosphoglycan), a galactofuranose-con taining glycoconjugate isolated from epimastigotes of T. cruzi was then employed as substrate. Galactopyranose, the product of the enzymatic activity, was detected by high pH anion-exchange chromatography, suggesting the presence of &#946;-D-galactofuranosidase in T. cruzi.

Tunicamycin nucleoside antibiotics were the first known to target the formation of peptidoglycan precursor lipid I in bacterial cell wall biosynthesis. They have also been used extensively as inhibitors of protein N-glycosylation in eukaryotes, blocking the biogenesis of early intermediate dolichyl-pyrophosphoryl-N-acetylglucosamine. Despite their unusual structures and useful biological properties, little is known about their biosynthesis. Elucidating the metabolic pathway of tunicamycins and gaining an understanding of the enzymes involved in key bond forming processes would not only be of great academic value in itself, it would also unlock a comprehensive toolbox of biosynthetic machinery for the production of tunicamycin analogues which have the potential to act as novel therapeutic antibiotics or as specific inhibitors of medicinally important NDP-dependent glycosyltransferases. I – Cloning the tunicamycin biosynthetic gene cluster. We report identification of the tunicamycin biosynthetic genes in Streptomyces chartreusis following genome sequencing and a chemically-guided strategy for in silico genome mining that allowed rapid identification and unification of an operon fractured across contigs. Heterologous expression established a likely minimal gene set necessary for antibiotic production, from which a detailed metabolic pathway for tunicamycin biosynthesis is proposed. II – Natural product isolation and degradation. We have developed efficient methods for the isolation of tunicamycins from liquid culture in preparative quantities. A subsequent relay synthesis furnished advanced biosynthetic intermediates for use as precursors in the production of tunicamycin analogues and as substrates for the in vitro characterisation of individual Tun enzymes. III – Functional characterisation of tun gene products. Individual tun gene products were over-expressed and purified from recombinant E. coli hosts, allowing in vitro functional studies to take place. An NMR assay of biosynthetic enzyme TunF showed it acted as a UDP-GlcNAc-4-epimerase. Putative glycosyltransferase TunD showed hydrolytic activity towards substrate UDP-GlcNAc but failed to accept to the expected natural acceptor substrate, providing unexpected insights into the ordering of biosynthetic events in the tunicamycin pathway. Initial studies into the over-expression of the putative sugar N-deacetylase TunE were also described. IV – Towards synthesis of tunicamycin fragments. Investigations into a novel synthesis of D-galactosamine – a structural motif within tunicamycin – led to the unexpected observation of inverted regioselectivity upon RhII-catalysed C-H insertion of a D-mannose-derived sulfamate. This was the first example of N-insertion at the beta- rather than gamma-C-H based on conformation alone and warranted further investigation. The X-ray structure of a key sulfamate precursor offered valuable insights as to the source of this unique selectivity.

Natural product drug discovery has traditionally been the corner stone of medicine having provided cures to many of today's most common diseases. In particular, antibiotics have revolutionised healthcare and extended human lifespan. However, since their introduction into the clinic, resistance to these drugs has arisen. With the number of new antibiotics being discovered in recent years declining, and fewer drugs making it past clinical trials, we have reached the point where antibiotic resistant infections have become common place and a serious threat to health and society. There is now an urgent requirement for the discovery of new antibiotics and in particular those with unexploited mode of action. This thesis details the different areas of natural product drug development from discovery through to analogue generation. In Chapter one, the history of natural products as therapeutics is explored with a particular focus on antibiotics and how resistance arises against these agents. It outlines why the discovery of new antibiotics is so important and new methods used to facilitate this search. Chapter two follows with the development of a screening platform for antibiotic induction, using the model Streptomyces; Streptomyces coleiolor M145. A variety of culture additives are explored for their ability to induce secondary metabolism production. Chapter three then details the sampling and identification of microbes from a pseudo-marine environment and their screening for their ability to produce secondary metabolites with antibiotic properties. The second half of this thesis centres on the non-ribosomal peptide echinomycin. Collaborators Aquapharm supplied the marine derived strain AQP-4895, capable of producing echinomycin. Chapter four details the establishment of AQP-4895 culturing conditions and the shift observed in production profile. Next Chapter five looks at producing echinomycin analogues through precursor directed biosynthesis. A range of halogenated quinoxaline carboxylic acids are synthesised and fed to AQP-4895, and the respective echinomycin analogues monitored by LC-MS. Chapter Six then aims to direct biosynthesis of the halogenated analogues, using mutasynthesis. Due to the lack of genetic data available surrounding the strain, an unusual approach was taken, using iPCR to create a template for homologous recombination.

Pilocarpus pennatifolius Lemmaire (Rutaceae) é uma espécie nativa que ocorre nas regiões Sul, Sudeste, Centro-Oeste e Nordeste do Brasil, e é popularmente conhecida por jaborandi. Essa e outras espécies de Pilocarpus encontram-se em risco de extinção devido ao extrativismo (uso como planta medicinal) e ao desmatamento. O cultivo in vitro de P. pennatifolius foi realizado como uma alternativa para a produção de pilocarpina (princípio ativo do jaborandi). A cultura de células foi obtida em meio MS semi-sólido, suplementado com sacarose, como fonte de carbono, e tendo como reguladores de crescimento BAP, IAA e NAA. O tempo de duplicação da cultura de calos foi de 22 dias. A concentração do alcalóide, determinada por técnicas cromatográficas, foi de aproximadamente 0,1 &#181;g/ mg de biomassa. Os ensaios enzimáticos referentes à biossíntese da pilocarpina tiveram como objetivo elucidar a etapa inicial de formação do anel imidazólico, além da localização desta reação na planta. Os experimentos realizados indicaram a presença da enzima histidina amino-transferase (HAT, EC 2.6.1.38) cuja atividade catalítica foi de 46,09 nKat/ mg de proteína, apenas no extrato protéico das raízes de P. pennatifolius. A determinação da composição do óleo essencial por CG e CG/EM indicou como constituintes majoritários os hidrocarbonetos tridecano (56,8 %) e pentadecano (25,5 %). Os sesquiterpenos não oxigenados constituíram cerca de 15 % do óleo obtido. / Pilocarpus pennatifolius Lemmaire (Rutaceae) is a native Brazilian species which is commonly known as jaborandi. This species is endangered due to its exploitation as medicinal plant and the increasingly deforestation. A callus culture of P. pennatifolius was established as an alternative source for pilocarpine production (the active compound of jaborandi). The cell culture was obtained in a semi-solid media, supplemented with sucrose as carbon source and as growth regulators, BAP, IAA e NAA. The doubling time for the callus culture was determined as 22 days. The concentration of the alkaloid, obtained by chromatographic techniques was ca. 0.1 &#181;g/ mg dry weight. The enzymatic assays related to pilocarpine biosynthesis were carried out with the aim to elucidate the imidazole ring formation beyond identify the site were this reaction takes place. The results indicated the presence of the enzyme histidine ammonia transferase (HAT, EC 2.6.1.38) only in the protein extract obtained from the roots of P. pennatifolius. The catalytic activity for this enzyme was 46.09 nKat/ mg protein. Volatile constituents from the leaves were analyzed by GC and GC/MS and the major compounds were determined as the hydrocarbons tridecane (56.8 %) and pentadecane (25.5 %). Almost 15 % of the total composition was constituted of non-oxygenated sesquiterpenes.