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The appended sugar residues of many natural products from Actinomyces are important for their biological activities. Many of these unusual sugar biosynthetic gene clusters have been isolated and many glycosyltransferases from various antibiotic-producing organisms have been identified. The increasing knowledge about these sugar biosynthetic pathways opens up the possibility of generating novel bioactive glycosylated compounds through combinatorial biosynthesis. The work described in this dissertation focuses on the investigation of the biosynthetic pathway of a rare nitro-containing sugar, D-kijanose, from an antibiotic, kijanimicin, and the glycosyltransferase helper proteins involved in the glycosylation of macrolide antibiotics. D-Kijanose, especially its nitro group, plays an important role in conferring the biological activities of the parent antibiotics. Cloning and sequencing of the kijanimicin biosynthetic gene cluster have allowed the proposal of the biosynthetic pathway of D-kijanose. The functions of the enzymes encoded by each open-reading frame in the cluster were also assigned based on sequence comparison with known enzymes found in other biosynthetic reactions. In this thesis, the functions of KijB1, a TDP-4-keto-6-deoxy-hexose 2,3-dehydratase, and KijD2, a TDP-hexose C-3 aminotransferase, were verified. The TDP-3-amino-4-keto-2,3,6-trideoxyhexose produced as an intermediate in the early stage of D-kijanose biosynthesis was also identified. In the second part of this dissertation, the in vivo protein-protein interaction between D-desosaminyl glycosyltransferase, DesVII, and its auxiliary protein, DesVIII, was established by yeast two-hybrid assay. The complex formation between these two proteins was also demonstrated by in vitro binding assay. Several strategies were tried to overexpress the D-mycaminosyl glycosyltransferase and its auxiliary protein, TylM2 and TylM3, although none of them were successful. A two-plasmid in vivo glycosylation system was also developed to test the competence of various DesVIII homologues to serve as the helper protein for glycosyltransferase DesVII, MycB and NbmD. In summary, the work in this dissertation has provided important information on the biosynthesis of D-kijanose and also significant insight into the function of the helper proteins of macrolide glycosyltransferases. These results could be useful for future studies of natural product biosynthesis and exploitation of glycodiversification. / text

Lavenders (Lavandula) are widely grown for their essential oils, which have extensive applications in cosmetics, hygiene products and alternative medicine. The therapeutic and olfactory properties of lavender essential oils are attributed to monoterpenes, a class of low molecular weight (C₁₀) isoprenoids. Oil composition in these plants is primarily determined by plant genotype, but can also be influenced by developmental and environmental factors. In order to define some of the mechanisms that control monoterpene abundance in lavenders, I measured the abundance of quality-defining monoterpenes in several L. angustifolia and L. x intermedia cultivars grown in the Okanagan. Data obtained confirmed that essential oil yield, as well as the abundance of camphor, borneol, linalool, and limonene was species-specific. L. angustifolia cultivars contained high amounts of linalool but yielded little oil, whereas L. x intermedia cultivars were rich in camphor and total oil. Monoterpene abundance changed during flower development, and differed between vegetative and reproductive tissues indicating differential regulation of the biosynthetic pathways, or specialized ecological functions. The abundance of linalool correlated with the transcription of the linalool synthase gene, suggesting that linalool production is in part regulated transcriptionally. However, the degree of correlation between linalool abundance and linalool synthase transcription differed between L. angustifolia and L. x intermedia, suggesting additional, and differing mechanisms that control linalool abundance in these species. In addition, monoterpene abundances were subject to loss during storage and suboptimal detection, two factors that must be considered in future analyses. Results obtained in this study provide insight into the regulation of monoterpene production in lavenders, and build the basis for future research aimed at improving essential oil production in these plants.

Lavenders (Lavandula) are widely grown for their essential oils, which have extensive applications in cosmetics, hygiene products and alternative medicine. The therapeutic and olfactory properties of lavender essential oils are attributed to monoterpenes, a class of low molecular weight (C₁₀) isoprenoids. Oil composition in these plants is primarily determined by plant genotype, but can also be influenced by developmental and environmental factors. In order to define some of the mechanisms that control monoterpene abundance in lavenders, I measured the abundance of quality-defining monoterpenes in several L. angustifolia and L. x intermedia cultivars grown in the Okanagan. Data obtained confirmed that essential oil yield, as well as the abundance of camphor, borneol, linalool, and limonene was species-specific. L. angustifolia cultivars contained high amounts of linalool but yielded little oil, whereas L. x intermedia cultivars were rich in camphor and total oil. Monoterpene abundance changed during flower development, and differed between vegetative and reproductive tissues indicating differential regulation of the biosynthetic pathways, or specialized ecological functions. The abundance of linalool correlated with the transcription of the linalool synthase gene, suggesting that linalool production is in part regulated transcriptionally. However, the degree of correlation between linalool abundance and linalool synthase transcription differed between L. angustifolia and L. x intermedia, suggesting additional, and differing mechanisms that control linalool abundance in these species. In addition, monoterpene abundances were subject to loss during storage and suboptimal detection, two factors that must be considered in future analyses. Results obtained in this study provide insight into the regulation of monoterpene production in lavenders, and build the basis for future research aimed at improving essential oil production in these plants.

Lavenders (Lavandula) are widely grown for their essential oils, which have extensive applications in cosmetics, hygiene products and alternative medicine. The therapeutic and olfactory properties of lavender essential oils are attributed to monoterpenes, a class of low molecular weight (C₁₀) isoprenoids. Oil composition in these plants is primarily determined by plant genotype, but can also be influenced by developmental and environmental factors. In order to define some of the mechanisms that control monoterpene abundance in lavenders, I measured the abundance of quality-defining monoterpenes in several L. angustifolia and L. x intermedia cultivars grown in the Okanagan. Data obtained confirmed that essential oil yield, as well as the abundance of camphor, borneol, linalool, and limonene was species-specific. L. angustifolia cultivars contained high amounts of linalool but yielded little oil, whereas L. x intermedia cultivars were rich in camphor and total oil. Monoterpene abundance changed during flower development, and differed between vegetative and reproductive tissues indicating differential regulation of the biosynthetic pathways, or specialized ecological functions. The abundance of linalool correlated with the transcription of the linalool synthase gene, suggesting that linalool production is in part regulated transcriptionally. However, the degree of correlation between linalool abundance and linalool synthase transcription differed between L. angustifolia and L. x intermedia, suggesting additional, and differing mechanisms that control linalool abundance in these species. In addition, monoterpene abundances were subject to loss during storage and suboptimal detection, two factors that must be considered in future analyses. Results obtained in this study provide insight into the regulation of monoterpene production in lavenders, and build the basis for future research aimed at improving essential oil production in these plants. / Graduate Studies, College of (Okanagan) / Graduate

The diversity of biosynthetic pathways in prokaryotes and eukaryotes has led to numerous bioactive natural products (NPs) which occupy a vast chemical space. Despite the current challenges in NP research, these molecules are still relevant today as they are a major source of human medicine as well as being useful biological tools. The elucidation of their biosynthetic pathways has also provided information about the biochemical and biophysical properties of fascinating enzyme families such as the α- oxoamine synthases (AOSs). The AOSs are an expanding group of pyridoxal 5’- phosphate (PLP)-dependent enzymes, which are involved in the biosynthesis of several important NP, including those essential for life. This study reports the characterization and structural analysis of a unique AOS denoted as TamD from Pseudoalteromonas tunicata. This enzyme plays a major role in tambjamine biosynthesis and consists of both an acyl carrier protein (ACP) domain and a PLP-binding catalytic domain. UV/vis spectroscopy and mass spectrometry (MS) of the recombinant TamD purified from E. coli revealed that the enzyme forms a Schiff base with PLP via Lys380, which is responsible for its characteristic yellow colour. It binds L-serine as a natural substrate with a Kd of 5.01 ± 0.64 mM. This thesis also reports structural data for TamD from xray crystallography at a resolution of 4.98 Å, which shows four molecules in the asymmetric unit (ASU) suggesting the enzyme exist as a dimer. The absence of the Nterminal region where the ACP domain is located in the crystal strucuture also suggests intrinsic flexibility and disorder within that region. With the increasing demand for new anti-infective therapies, investigations of the molecular interactions between NPs and their protein targets are vital in understanding the inhibition or activation properties of these molecules. The cysteine transpeptidases known as sortases produced by Gram positive bacteria have been identified as attractive targets for NP inhibitors. In this thesis, the molecular basis for the inhibition of Streptococcus mutans sortase A (SrtA) by the plant flavonoid, trans-chalcone is explored, using a combination of MS, enzyme kinetics, molecular modelling and x-ray crystallography. This study reports the first high resolution crystal structure of the H139A mutant of S. mutans SrtA, which reveals a unique N-terminal α-helix domain. Trans-chalcone was found to inhibit the in vitro activity of S. mutans SrtA in a slow, tight–binding manner, with a half maximal inhibitory concentration (IC50) of 5.0 ± 0.6 μM. The interaction resulted in a covalent adduct with the active site cysteine residue (Cys205) via a Michael addition mechanism. Additionally, trans-chalcone showed evidence of S. mutans anti-biofilm activity in a concentration dependent manner up to 250 μM with an efficacy cut-off point at higher concentations. These results indicate that chalcone flavonoids are worth further investigation as potential antibiofilm inhibitors. A renewed interest in plant NPs has also led to a collaborative investigation on the antimicrobial potential of garlic-derived allicin, against Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. Allicin is the principal antibacterial agent in fresh preparations of garlic extracts. This investigation reports the first evidence that allicin and allicin-contaning garlic extracts possess inhibitory and bactericidal activities against Bcc. The minimum inhibitory concentrations (MICs) of aqueous garlic extract (AGE) against 38 Bcc isolates ranged from 0.5 to 3% (v/v). An investigation into the possible molecular mechanisms of allicin with a recombinant thiol-dependent peroxiredoxin (BCP) from B. cenocepacia revealed that allicin and AGE modify an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit further investigation as adjuncts to existing antibiotics.

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