The purpose of this study was to investigate whether increased endogenous cholesterol synthesis contributes to the elevated plasma cholesterol levels observed in type III hyperlipoproteinemia (type III HLP). Eight apolipoprotein (apo) E2 subjects with type III HLP and 8 apo E2 non-hyperlipidemic control subjects (controls) were given a priming bolus dose of deuterium oxide (D₂O) (0.7 g D₂O/kg body H2O). Daily M1 (central) pool free cholesterol fractional synthetic rate (FSR) was calculated as the incorporation rate of deuterium from body water into plasma free cholesterol. Blood samples were collected one half hour prior to, and at 12 hour intervals over 48 hours following, the bolus D₂O dose. Drinking water labelled at 1.4 and 0.7 g D₂O/liter H₂O was given on the fed and fasted days, respectively. Over 0-24 hours, subjects consumed a diet of three isocaloric meals which, in composition, approximated average North American intakes. Subjects fasted over 24-48 hours. The deuterium enrichment of plasma free cholesterol and plasma water was determined by isotope ratio mass spectrometry. When all subjects were included, mean (±SEM) free cholesterol overall FSR in type III HLPs (0.031 ± 0.006 per day) was not significantly different from controls (0.037 ± 0.004 per day). Estimated Ml total cholesterol pool size in type III HLPs (26.1 ± 1.9 g) and controls (24.9 ± 0.6 g) was not significantly different. When free cholesterol net synthesis was calculated as the absolute amount of cholesterol synthesized per day, based on Ml total cholesterol pool size, overall free cholesterol net synthesis in type III HLPs (0.304 ± 0.034 g/day) was not significantly different from controls (0.364 ± 0.035 g/day). When all subjects were included, overall free cholesterol FSR and overall free cholesterol net synthesis were significantly greater (p<0.001) in the fed (0.066 ± 0.006 day⁻¹ and 0.655 + 0.048 g/day, respectively) as compared to the fasted state (0.001 ± 0.004 day⁻¹ and 0.010 ± 0.037 g/day, respectively). In the fed state, type III HLPs tended to synthesize cholesterol at a lower rate and in a lower absolute amount as compared to controls, while the reverse was observed in the fasted state. These results suggest that: (1) the elevated plasma cholesterol levels observed in type III HLPs are not due to excess de novo cholesterol synthesis; (2) fasting significantly reduces cholesterol synthesis from the fed state. / Land and Food Systems, Faculty of / Graduate

Pseudomonas aeruginosa, a ubiquitous environmental bacterium and an opportunistic human pathogen, forms biofilms through a series of interactions between the cells and adherence to surfaces. Not only does rhamnolipid contribute to the pathogenic potential of P. aeruginosa, but it has also been reported that the bacterium utilises rhamnolipid to actively maintain the void spaces surrounding microcolonies, thus contributing to the architecture of P. aeruginosa biofilms. The P. aeruginosa rhlAB operon encodes the enzyme rhamnosyltransferase I, which produces mono-rhamnolipid, and the induction of rhlAB is dependent on the quorum sensing transcription activator RhIR complexed with the auto inducer N-butyryl-homoserine lactone. In this study, several aspects related to rhamnolipid biosynthesis and regulation in P. aeruginosa PAO1 were investigated. As a first step, a biochemical assay was developed and optimised whereby the concentration of rhamnolipid could be accurately quantified following its extraction from small sample volumes. Although the optimised rhamnolipid assay is not able to distinguish between different rhamnolipids or between different homologs of a specific rhamnolipid, it is, however, simple to perform, cost¬effective and does not rely on the use of specialised equipment. Subsequently an rhlAB-deficient mutant strain of P. aeruginosa PAOI strain was generated. For this purpose, three allelic exchange strategies, i.e. plasmid incompatibility, the use of a SacB counter-selectable marker and a combination of these approaches, were investigated by making use of newly constructed allelic exchange vector systems. The results that were obtained indicated that, of the three approaches, the latter was most efficient in generating the desired P. aeruginosa mutant strain, and 90% of the derived strains were found to be double reciprocal mutants. Reporter gene technology, using the genes encoding for stable and unstable variants of the green fluorescent protein (GFP), was finally used to investigate the transcriptional activity of the rhlA promoter in P. aeruginosa biofilms under conditions of continuous flow using glass as substratum. For this purpose, mini-CTX-GFP reporter vectors, containing stable and unstable variants of the gfp reporter gene, were constructed that allow for integration of a single copy of the transcriptional fusion in a defined, non-essential region onto the P. aeruginosa genome. Several global regulators have been reported to playa role in regulating quorum sensing and/or rhamnolipid biosynthesis in P. aeruginosa, amongst other, the sigma factors RpoS and RpoN. Therefore, rhlA promoter activity was also investigated in biofilms of P. aeruginosa strains lacking either RpoN or RpoS. Although structural differences between the biofilms formed by the P. aeruginosa wild-type PAD 1 and respective mutant strains were noted, transcription of rhlA appeared to be constitutive from 24 h onwards and did not appear to be localised to specific areas within the microcolonies or biofilms. These results, combined with those obtained by batch analysis, indicated that RpoS positively regulates rhlA transcription, whilst RpoN did not appear to influence rhlA promoter activity under the conditions used in this study. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

The 7-deazaguanosine nucleosides queuosine (Q) and archaeosine (G⁺) are two of the most structurally complex modified nucleosides found in tRNA. Q is found exclusively in the wobble position of tRNAGUN coding for the amino acids asparagine, aspartate, histidine and tyrosine in eukarya and bacteria, while (G⁺) occurs in nearly all archaeal tRNA at position 15. In archaea preQ₀ is inserted into tRNA by the enzyme tRNA-guanine transglycosylase (TGT), which catalyzes the exchange of guanine with preQ₀ to produce preQ₀-tRNA. The first objective of this study was to identify and characterize the enzyme(s) catalyzing the conversion of preQ₀-tRNA to G+-tRNA. Comparative genomics identified a protein family possibly involved in the final steps of archaeosine biosynthesis, which was annotated as TgtA2. Structure based alignments comparing TGT and TgtA2 revealed that TgtA2 lacked key TGT catalytic residues and contained an additional module. The gene corresponding to "tgtA2" from "Methanocaldococcus jannaschii (mj1022)" was cloned, expressed and the purified recombinant enzyme characterized. Recombinant MjTgtA2 was shown to convert preQ₀-tRNA to G⁺-tRNA using glutamine, asparagine or NH₄⁺ as nitrogen donors in an ATP-independent reaction. This is the only example of the conversion of a nitrile to a formamidine known in biology. QueF catalyzes the reduction of preQ₀ to 7-aminomethyl-7-deazaguanine preQ₀ in the queuosine biosynthetic pathway. The second aim of this study was the transient state kinetic analysis of substrate binding and catalysis by the enzyme QueF, as well as investigation of the effects of ligands on its quaternary structure. Gel filtration and sedimentation equilibrium analyses indicated that QueF exists as a hybrid population in a rapid equilibrium between decamer and pentamer states. Addition of preQ₀ to QueF resulted in shifting the equilibrium towards the decamer state, as did the addition of divalent metals. Potassium chloride at high concentrations was found to disrupt the quaternary structure of QueF. Intrinsic tryptophan and NADPH fluorescence was used to determine the substrate binding to QueF by stopped-flow kinetic studies. Studies on the binding of preQ₀ to QueF in conjuction with binding NADPH to the QueF mutant E78A-thioimide intermediate suggested a two-step mechanism consisting of a fast bimolecular process and a subsequent slower unimolecular process, while the binding of preQ₀ to the C55A mutant was monophasic, consisting of only the fast bimolecular process. Thioimide formation was monitored by UV-Vis; under single turnover conditions the data fit well to single exponential rise. However, at high preQ₀ concentrations two phases could be observed. The reduction of the thioimide was determined under single turnover conditions by both UV-Vis and fluorescence, and comparable rates were obtained from both techniques. These results indicate that the binding of preQ₀ and NADPH to QueF, as well as thioimide formation, are very rapid; and that reduction of the thioimide is most likely the rate limiting step. Analysis of component rates suggests structural changes occur between these steps, further limiting the overall rate.

<p> The biosynthesis of the alkaloids of Decodon verticillatus, (L.)Ell, a member of the Lythraceae family, was studied by tracer methods. These alkaloids contain a phenylquinolizidine system, whose biosynthetic origin has not previously been investigated.</p> <p> Labelled samples of lysine, △^1-piperideine and phenylalanine yielded radioactive decodine and decinine, the two major alkaloids of D. verticillatus. Systematic degradations of these labelled alkaloids show that lysine, △^1-piperideine and phenylalanine serve as specific precursors. Whereas lysine and △^1-piperideine entered the quinolizidine ring, the biphenyl ring system of these alkaloids was derived from two units of phenylalanine. In a further experiment, labelled pelletierine was administered to the plants. But this experiment yielded an inconclusive result.</p> / Thesis / Doctor of Philosophy (PhD)

<P> Taxol, a highly functionalized and complex diterpene belonging to the taxane group, possesses strong antitumor activity against various cancers, especially in cases of advanced ovarian and breast cancers. Because of the unique mechanism of action and the unusual chemical structure, taxol may represent the prototype of a new class of chemotherapeutic agents. The non-clinical work up to date on taxol is described in this thesis, including natural resources, chemical synthesis and chemical manipulation of taxol. </p> <p> The biosynthesis of taxol has not yet been studied. The hypothesis proposed for the biosynthetic sequence involves cyclization of geranylgeranyl pyrophosphate (GGPP) into hydrocarbon intermediates, which are then further transformed into taxol. In order to study the biosynthesis of taxol, [^14C]-labelled GGPP was prepared. Through the use of this labelled precursor in incubations with cell-free extract of yew labelled biosynthetic intermediates which are formed can be isolated and identified, hence leading to further understanding of the biosynthesis of taxol. </p> <p> The synthesis of [16,17-^14C]geranylgeranyl pyrophosphate was achieved in eight steps starting from commercially available geranylgeraniol. The alcohol was protected as the acetate derivative and the terminal double bond selectively epoxidized. The epoxide was opened to the diol, which was then cleaved. The resulting aldehyde was coupled to [^14C] isopropyltriphenylphosphonium ylid in a Wittig reaction, giving [16,17-14C]geranylgeraniol after deprotection of the acetate group. The alcohol was converted into the chloride derivative and subsequently to [16,17-14C]geranylgeranyl pyrophosphate. </p> / Thesis / Master of Science (MSc)

No description available.

The biosynthesis of cellulolytic enzymes by resting cells of T. reesei QM 9414 incubated in 17 mM potassium phosphate buffer, pH 6.0, and Q-β-D-glucopyranosy1-(172)α-D-glucopyranose was investigated. A maximum of 200 mi1liunits aryl-β-D-g1ucosidase, 9000 mi1liunits endo-l,4-β-D-glucanaseand 200 milliunits Avicelase per milliliter of culture supernate was produced after 24 hours of incubation; at that time, extracellular protein production reached a maximum of 0.5 mg/ml. Optimum enzyme yields were obtained with 3-4 mg dry weight cells/ml, and 1 nIDi 0-β-D-glucopyranosyl-(1->2)α-D-glucopyranose. Inclusion of metals, e.g., zinc, cobalt, manganous and ferrous ions enhanced enzyme production when glutamic acid was present, in which case aryl-β-D-glucosidase, endoglucanase and Avicelase activity were enhanced by 20, 100 and 40 percent, respectively. Under the same conditions asparagine enhanced only ary1-β-D-glucosidase activity. Some of the enzymic components of the cellulase system produced by T. reesei under these conditions were purified by ion exchange chromatography on DEAE-Sephadex A-50. Two cellobiohydrolases were isolated in pure form. One of these was identical to the previously isolated ce1lobiohydrolase D on the basis of amino acid composition carbohydrate content, electrophoretic mobility and ultraviolet spectrum. Similar data for the other cellobiohydrolase suggest that it represents an enzyme not previously identified. An endoglucanase was also isolated which, on the basis of its amino acid composition and electrophoretic mobility, appears similar to the previously identified Endoglucanase IV. More precise characterization of these enzymes is currently under investigation. / Master of Science

Antibiotics form the cornerstone of modern medicine, facilitating advancements in numerous healthcare fields and contributing to unprecedented increases in human life expectancy. However, the efficacy of these life-saving drugs is now jeopardized by the rise of antimicrobial resistance. This growing threat is exacerbated by the slow pace of new antibiotic discoveries. The drug discovery process is both time-consuming and costly, and efforts to identify novel antibiotics often result in the rediscovery of known antibiotics, further hindering progress. To combat antibiotic resistance and facilitate the discovery of novel drugs, several approaches can be employed. First, understanding the mechanisms of resistance found in environmental bacteria is crucial for preparing against the potential mobilization of these resistance mechanisms into pathogenic bacteria. Second, developing tools that make the drug discovery process less costly and time-consuming can accelerate the discovery rate and broaden participation in drug discovery efforts. Finally, understanding how bacteria synthesize natural product antibiotics provides invaluable information that can be leveraged in drug discovery efforts, including synthetic biology approaches. This thesis addresses efforts and challenges in the various aspects of drug discovery. To enhance our understanding of environmental resistance mechanisms, I conducted a screen for ciprofloxacin-inactivating enzymes and characterized the activity of a previously reported ciprofloxacin-inactivating enzyme, CrpP. These findings highlight the difficulties associated with discovering synthetic antibiotic-inactivating enzymes. To contribute to drug discovery, I expanded the Antibiotic Resistance Platform and developed a streamlined version to improve antibiotic dereplication efforts, thereby accelerating the natural product discovery process. Additionally, I investigated the mechanism of β-serine biosynthesis, a nonproteinogenic amino acid found in the antibiotic edeine. By elucidating how β-serine is synthesized, this information can be applied to synthetic biology approaches for drug discovery. / Thesis / Doctor of Philosophy (PhD) / Antibiotics used in medical treatments today often originate from natural sources like environmental bacteria and are known as natural product antibiotics. These natural product antibiotics are essential for treating bacterial infections and play a crucial role in modern medicine, including surgery and cancer treatment. However, the increasing problem of antimicrobial resistance and the lack of new drugs being discovered threatens the effectiveness of these life-saving medicines. To combat antibiotic resistance and protect the use of antibiotics, we need to understand how bacteria resist antibiotics, develop better methods for discovering new antibiotics, and gain insights into how bacteria produce natural product antibiotics. This thesis addresses these challenges by trying to find bacteria that can break down antibiotics, improving a tool for drug discovery, and understanding how bacteria make the antibiotic known as edeine. These efforts advance our understanding of antibiotic resistance and pave the way for developing new and effective antibiotics.

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

This thesis entitled ‘ Unusual Acylation Properties of Type II Fatty Acid Biosynthesis Acyl Carrier Proteins’ describes the discovery of self-acylation and malonyl transferase activity in acyl carrier proteins involved in type II fatty acid biosynthesis and assigns a physiological role to these processes inside the cellular milieu. Acyl carrier protein (ACP) is one of the most abundant proteins present inside the cell and almost 4% enzymes require it as a cofactor. Acyl carrier proteins can exist either as discrete proteins or as domains of large functional proteins. They function in a variety of synthases as central molecules to which growing acyl intermediates and nascent product molecules are covalently tethered during the elongation and modification steps required to produce the final product. A prototypical bacterial ACP is composed of 70-80 amino acids and is generally expressed in the apo form. It is post-translationally modified to active holo form by the addition of 4'-phosphopantetheine moiety to an absolutely conserved serine residue in a reaction catalyzed by holo-ACP synthase or 4'-phosphopantetheine transferase. Chapter 1 surveys literature related to carrier proteins inside the cell and describes the thesis objective. It also presents an overview of the acyl carrier proteins and their involvement in various metabolic pathways inside the cell. The chapter details the structural organization of acyl carrier proteins from various sources revealing the conservation in their structure and also details the molecular basis of interaction of ACP with other enzymes inside the cell. The discovery of unusual self-acylation property in acyl carrier proteins involved in polyketide biosynthesis and its absence in acyl carrier proteins involved in fatty acid biosynthesis prompted me to investigate the reasons for this selective behavior. Discovery of self-acylation property in acyl carrier proteins Plasmodium falciparum and chloroplast targeted Brassica napus acyl carrier proteins involved in type II fatty acid biosynthesis and the mechanism of this reaction forms the basis of Chapter 2. In this chapter it has been shown that self-acylation property is intrinsic to a given acyl carrier protein and is not dependent on the pathway in which it is involved. Based on primary sequence analysis and site directed mutagenesis studies presence of an aspartate/glutamate has been identified to be critical for the self-acylation event. Furthermore, it has also been shown that the self-acylation event in type II fatty acid biosynthesis acyl carrier proteins is highly specific in nature employing only dicarboxylic acid –CoAs as substrates unlike the polyketide biosynthesis acyl carrier proteins which utilize both dicarboxylic acid and β-keto acid thiol ester -CoAs as substrates. The detailed kinetics of these reactions has also been worked out. Combining all the results a plausible mechanism for the self-acylation reaction has been proposed. Chapter 3 describes the discovery of a novel malonyl transferase behavior in acyl carrier proteins involved in type II fatty acid biosynthesis. Malonyl transferase property in ACPs of type II FAS from a bacterium (Escherichia coli), a plant (Brassica napus) and a parasitic protozoon (Plasmodium falciparum) were investigated to present a unifying paradigm for the mechanism of malonyl transferase behavior in ACPs. Identification of malonyl transferase property in Plasmodium falciparum ACP and Escherichia coli ACP (EcACP) and the absence of this property in Brassica napus ACP has been described in this chapter. Detailed investigations demonstrated that presence of an arginine or a lysine in loop II and an arginine or glutamine at the start of helix III as the residues that are critical for the transferase activity. In order to assign a physiologic function to these unusual acylation properties, fabD(Ts) mutant strain of Escherichia coli was utilized for heterologous complementation by the various wild type and mutant ACPs that are able to catalyze either or both of the activities. Growth of the mutant strain at non-permissive temperature, when complemented with ACPs catalyzing both the reactions confirmed that these properties have a physiologic relevance. Extensive mutagenesis experiments in conjunction with complementation studies allowed me to propose a plausible mechanism on how the self-malonylation and malonyl transferase properties operate in tandem. Chapter 4 describes the thermodynamic characterization of self-acylation process using Isothermal Titration Calorimetry. Isothermal Titration Calorimetric studies on the binding of malonyl, succinyl, butyryl and methylmalonyl –CoA to Plasmodium falciparum and Brassica napus acyl carrier proteins were performed to investigate the role of thermodynamic parameters in the specificity of self-acylation reaction. Calculation of the parameters showed that the thermodynamics does not control the self-acylation reaction. The evolution of self-acylation property in various acyl carrier proteins and its possible significance in the evolution of various metabolic events is described in Chapter 5. Extensive bioinformatics search was performed and phylogenetic analysis on acyl carrier proteins from 60 different taxa was done using the MEGA4 program. Analysis showed that this property was first found in cyanobacterium. Later, during the course of evolution this property was lost in most acyl carrier proteins, and was retained either in acyl carrier proteins that are targeted to organelles of cyanobaterial orgin viz. apicoplast in apicomplexans and chlorplasts in plants or in acyl carrier proteins involved in secondary metabolic events such as polyketide biosynthesis. Chapter 6 summarizes the findings of the thesis. Acyl carrier protein from Plasmodium falciparum, Brassica napus and Escherichia coli were characterized for their self-acylation and malonyl transferase properties and a combined mechanism for these two properties is proposed. The work done also provides an in vivo rationale to these in vitro processes. Furthermore, the evolutionary significance of the self-acylation behavior is also discussed in the thesis. The thesis also probes into the thermodynamics of the self-acylation reaction in Plasmodium falciparum and Brassica napus acyl carrier proteins. Thus, the thesis adds a new dimension to the much unexplored ACP biology and paves the way to study in vivo roles of these processes in detail. Appendix I describes the Isothermal Titration calorimetric characterization of binding of various acyl-PO4 molecules to Escherichia coli PlsX (Acyl-phosphate acyltransferase). PlsX, the first enzyme of phosphatidic acid biosynthesis pathway catalyzes the conversion of acyl-ACP into acyl-PO4, which is further used by other enzymes leading to the formation of phosphatidic acid. ITC results presented in this section show that longer chain length acyl-PO4 molecules show better binding to PlsX, as compared to the smaller ones demonstrating that long chain acyl molecules serve as better substrates for phosphatidic acid synthesis.