Thesis (Ph. D.)--University of Hong Kong.

Thesis (M. Sc.)--University of Hong Kong, 1969. / Typewritten.

Microalgae have been proposed as a potential feedstock for biofuel production, and as a result, interest in the biology of these organisms has intensified. These organisms also synthesize lipids that are vital to human health and nutrition. Stress has been shown to have an effect on lipid composition and gene expression in microalgae, but many studies have focused on the effects of abiotic stressors. The purpose of this study was to investigate the effect of biotic stress on lipid-related gene expression in Phaeodactylum tricornutum, a model species of microalgae. The source of biotic stress used in this study was 2E,4E/Z-decadienal, a diatom-derived oxylipin that has been shown to function as a stress signal among diatoms. Real-time RT-qPCR analysis revealed that expression of a patatin-like phospholipase was significantly decreased in decadienal-treated cultures as compared to a solvent control. The expression of a delta-9 desaturase gene believed to be responsible for production of 16:1 fatty acids was increased by a factor of 12. FabI, a gene involved in fatty acid biosynthesis, and PtD5a, which codes for an ER-localized desaturase, were both down-regulated in cells exposed to decadienal. However, changes in expression were only shown to be significant for the patatin-like phospholipase gene. Increased expression of the delta-9 desaturase gene may be a protective mechanism against infection from pathogens, since 16:1 fatty acids have been shown to have antibacterial properties. Regulation of membrane desaturation may also serve to stabilize photosynthetic membranes during times of stress. The down-regulation of the phospholipase gene was surprising, since the release of fatty acids from membrane lipids for oxylipin production is a common response to stress. It is recommended that this experiment be improved upon and expanded in order to determine whether the results obtained are reproducible and how these changes in gene expression correlate with physiological effects. / text

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The focus of this dissertation is the study of two enzymes, DXR and HppE. DXR catalyzes the first committed step in the MEP pathway, which is the pathway most eubacteria, archeabacteria, algae, and the plastids of plants use for the biosynthesis of isoprenoid. Since mammals utilize the mevalonate pathway and isoprenoids are essential for survival, all enzymes in the MEP pathway are excellent antibiotic targets. One antibiotic that has promise in the fight against malaria is the natural product fosmidomycin, whose antibiotic activity is due to its ability to bind and inhibit DXR. With a deeper understanding of DXR's catalyzed reaction, it will be possible to design a more sophisticated and potent antibiotic. To probe the mechanism of DXR, two fluorinated substrate analogues, 3F-DXP and 4F-DXP, and a fluorinated product analogue, FCH₂-MEP were designed and analyzed as possible substrates or inhibitors. To further analyze the mechanism of DXR, a 2° [²H]-KIE study was conducted using the equilibrium perturbation method. The second enzyme this dissertation examines is HppE, which catalyzes the final step in the biosynthesis of the antibiotic, fosfomycin. Fosfomycin is a clinically useful antibiotic for the treatment of limb-threatening diabetic foot infections and urinary tract infections. Chemically speaking, HppE is unique for two reasons. First, HppE's epoxidation differs from Nature's standard method of epoxide formation by alkene oxidation, where the epoxide oxygen is derived from molecular oxygen. For HppE, the epoxide is formed through the dehydrogenation of a secondary alcohol; thus the epoxide oxygen is derived from the substrate. Second, HppE is a unique member of the mononuclear non-heme iron-dependent family of enzymes. HppE differs from all other mononuclear non-heme iron-dependent enzymes by requiring NADH and an external electron mediator for turnover but not requiring [alpha]-KG, pterin, ascorbate, or an internal iron-sulfur cluster. After a study was published on the activity of zinc-reconstituted HppE from Streptomyces wedmorensis, the proposed iron and NADH dependent mechanism of HppE was reevaluated and was reconfirmed. The HppE from Pseudomonas syringae (Ps-HppE) was also purified and was characterized biochemically and spectroscopically. The results of [²H] and [¹⁸O]-KIE studies on Ps-HppE are also reported. / text

α-Isopropylmalate synthase (α-IPMS) is responsible for catalysing the first committed step in leucine biosynthesis. This pathway is found in plants and microorganisms, including pathogenic bacteria such as Mycobacterium tuberculosis and Neisseria meningitidis. α-IPMS catalyses a Claisen condensation reaction between α-ketoisovalerate (KIV) and acetyl coenzyme A (AcCoA) to form the product α-isopropylmalate (IPM). This enzyme undergoes feedback inhibition by the end product of the pathway, leucine. This regulation allows the control of the rate leucine biosynthesis. This project focuses on the α-IPMS enzymes from M. tuberculosis and N. meningitidis (MtuIPMS and NmeIPMS). These α-IPMS enzymes are homodimeric in structure. Each monomer consists of a catalytic domain which comprises of a (β/α)8 barrel fold, two subdomains and a regulatory domain, to which the allosteric binding of the natural inhibitor leucine occurs. The mechanism by which the allosteric binding of leucine leads to a decrease in enzymatic activity is not yet fully understood. Citramalate synthase (CMS) is responsible for catalysing the first committed step of threonine-independent isoleucine biosynthesis. This enzyme is extremely similar to α-IPMS in both the reaction which it catalyses and the catalytic and regulatory domain structure. CMS catalyses a Claisen condensation reaction between pyruvate and AcCoA to produce citramalate (CM). CMS is also feedback inhibited by the end product of its pathway, isoleucine. The similarity between α-IPMS and CMS enzymes resulted in and examination of the inhibitor selectivity of MtuIPMS. Amino acids in the leucine binding site were altered to their counterparts in the isoleucine binding site of the CMS enzyme to see if the selectivity of the leucine binding site could be interchanged. Results from this study show that it is possible to change inhibitor selectivity with a single amino acid substitution. However, changing the selectivity from leucine to isoleucine was unsuccessful. Instead, one of the MtuIPMS variants displayed significantly increased sensitivity to an alternative amino acid, norvaline. The MtuIPMS variants were expressed and purified using immobilised metal affinity chromatography and size-exclusion chromatography. These variants were then kinetically characterised and displayed similar binding affinities and turnover rates for the natural substrates to the wild-type enzyme. As expected changes to the leucine binding pocket had drastic effects on the sensitivity of the enzyme to its natural inhibitor. This work is described in Chapter 2 of this thesis. The mechanism by which the regulatory signal is transferred from the allosteric leucine binding site to the catalytic site in α-IPMS is not fully understood. NmeIPMS variants were created based on preliminary molecular dynamic simulations which indicated that significant changes in residue contacts were associated with leucine binding. Chapter 3 describes studies that explore the effect of single amino acid substitutions of NmeIPMS. The NmeIPMS variants were expressed and purified similarly to MtuIPMS, using immobilised metal affinity chromatography and size-exclusion chromatography. Variants were subsequently characterised via mass spectrometry, differential scanning fluorimetry and kinetic assays. It was found that each variant generated retained sensitivity to leucine but displayed significant differences in the catalytic efficiencies with AcCoA. One of the generated variants also displayed a significant increase in thermal stability. Results are drawn together in Chapter 4 along with future directions of this research. This chapter details knowledge gained into protein structure and allosteric mechanisms in this thesis.

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N-glycosylation is a metabolic process found in all three domains of life. It is the attachment of a polysaccharide glycan to asparagine (Asn) residues within the amino acid motif, Asn-Xaa-Ser/Thr. In the archaeon, Methanococcus maripaludis, a tetrasaccharide glycan was isolated from purified flagella and its structure determined by mass spectrometry analysis. The linking sugar to the protein is surprisingly, N-acetylgalactosamine (β-GalNAc), with the next proximal sugar a derivative of N-acetylglucosamine (β-GlcNAc), being named β-GlcNAc3Ac, and the third sugar a derivative of N-acetylmannosamine (β-ManNAc), with an attached threonine residue on the C6 carbon (β-ManNAc3NAm). The terminal sugar is an unusual diglycoside of aldulose ((5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose). Previous genetic analyses identified the glycosyltransferases (GTs) responsible for the transfer of the second and third sugars of the glycan, as well as the oligosaccharyltransferase (OST) which attaches the glycan to protein. Left unidentified were the first and fourth GTs, the flippase as well as any genes involved in glycan sugar biosynthesis and modification. In this work, genes suspected to be involved in the biosynthesis of N-linked sugars, as well as those that might encode the missing GTs and flippase were targeted for in-frame deletion. Mutants with a deleted annotated GT gene (MMP1088) had a small decrease in flagellin molecular weight as determined by immunoblotting. Mass spectrometry (MS) analysis confirmed that the N-linked glycan was missing the terminal sugar as well as the threonine found on the third sugar of wildtype cells. Mutants with a deleted gene annotated to be involved in acetamidino synthesis (a functional group that is present on the third sugar), also had a decrease in flagellin molecular weight. MS analysis determined that the N-linked glycan was missing the acetamidino group on the third sugar as well as its attached threonine, along with the terminal sugar. Both mutants were able to assemble functional flagella but had impaired motility compared to wildtype cells in mini-swarm agar. Deletions were also constructed in four other GT genes considered candidates in assembly of the linking sugar. However, none of these mutants had the expected decrease in flagellin molecular weight. With the work done in this study, the glycosyl transferase that attaches the last sugar of the M. maripaludis N-linked assembly pathway has been identified as well as a gene involved in the biosynthesis and modification of the glycan sugars. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-07-07 15:45:19.052

Loline alkaloids (LA) are secondary metabolites produced by Epichloandamp;euml; (anamorph, Neotyphodium) grass endophytes. They are toxic and deterrent to a broad range of herbivorous insects but not to livestock. This protective bioactivity has spurred considerable research into the LA biosynthetic pathway. LOL, the gene cluster containing nine genes, is required for LA biosynthesis. The regulation of LOL genes during LA production in culture and in symbio is of interest. In this study, coordinate regulation between LOL gene expression and LA production level was investigated in both MM culture and symbiota. Results showed that expression of LOL genes in N. uncinatum MM culture were tightly correlated with each other (p andamp;lt; 0.0005), and all presented a significant temporal quadratic pattern during LA production. Gene expression started before LA were detectable, and increased while LA accumulated. The highest gene expression level was reached before the highest amounts of LA were detected, and gene expression level declined to a very low level after amounts of LA plateaued. Observations suggested that the hierarchical clusters based on the correlation coefficient could help to predict the roles of LOL genes in the LA pathway. In symbiota, coordinate coregulation of LOL gene expression with LA was found in E. festucae-meadow fescue inflorescences and stromata, whereby lower LOL gene expression corresponded with the lower LA level in stromata. In N. uncinatum (or N. siegelii)-meadow fescue vegetative tissues, dramatically higher LA levels were found in younger leaf tissue than in older leaf tissue, yet no evidence was found to relate this difference to LOL gene expression differences. Instead, substrate availability may regulate the LA level. In particular, asparagine was more than 10-fold higher in young leaf tissue than in old tissue, although proline was significantly lower in young tissue. Therefore, different regulatory mechanisms underlie LOL gene expression and LA production in different circumstances. The GUS activity of Pro-lolC2-GUS and Pro-lolA2-GUS in Neotyphodium species was almost undetectable in culture, though the activity could be detected in symbiota. The mRNA of GUS did not exhibit the same pattern as lolC2 or lolA2 in culture during LA production time course. A Pro-lolC2-cre transgene was expressed in complex medium, in which lolC2 mRNA was not detectable. These results suggest that proper regulation of LOL genes in culture or symbiota is dependent on the LOL cluster.