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<p>Nonribosomal peptides (NRPs) are a major class of natural products known for their biological activities and are employed therapeutically as immunosupressants, anticancer agents, and antibiotics. Nonribosomal peptides are microbial products, biosynthesized by large assembly line-like enzymes, known as nonribosomal peptide synthetases (NRPSs) that can be found in large gene clusters within the genome. With the advent of genome sequencing, the gene clusters for known NRPs are easily identified within producing organisms, but more strikingly, this sequencing reveals that microbes often contain many gene clusters with no known products suggesting traditional methods of isolation are overlooking the majority of NRPs.</p> <p>Extensive studies of NRPS functions have revealed assembly line logic for the biosynthesis of NRPs and using this knowledge, the NRP products of NRPS gene clusters can be predicted. In this research, products from both a simple dimodular NRPS from <em>Staphylococcus aureus </em>and a complex 11 module NRPS from <em>Delftia acidovorans </em>were predicted and used to successfully identify and isolate two novel NRPs, aureusimine and delftibactin.<em> </em>Theses compounds fell outside traditional NRP activities, one being a virulence regulator and the other a gold-complexing metallophore. Subsequent biosynthetic studies of the aureusimine gene cluster within the heterologous host, <em>Escherichia coli</em>, provide insight into NRPS flexibility for the creation of NRP natural variants and highlighted the utility of <em>E. coli </em>for the heterologous production of NRPs.</p> <p>Realizing single NRP predictions are not always accurate, a strategy was devised to use a genomically predicted NRP fragment barcode databases with the LC-MS/MS dereplication algorithm, iSNAP, to chemoinformatically identify and physically locate genetically predicted NRPs within crude extracts. This final contribution eliminates the need for bioactivity guided approaches to discovery and provides a strategy to systematically discover all predicted NRPs from cryptic gene clusters. This thesis delivers strategies and technologies for the directed discovery of NRPs from microbial sources.</p> / Doctor of Philosophy (PhD)

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Through the ICBG (International Cooperative Biodiversity Group) program and a continuing search for anticancer compounds, plant extracts were obtianed from Suriname and Madagascar and screened for cytotoxic activity in the A2780 human ovarian cancer cell line. Fractionation of a leaf and flower extract of Casearia nigrescens led to the isolation of six new clerodane diterpenes. Four were new natural products and the other two were previously unreported hydrolysis products. Their structures were determined using mass spectrometry and 1-D and 2-D NMR. All six compounds were cytotoxic in the A2780 human ovarian cancer cell line. Fractionation of a leaf extract of Vernonia pachyclada led to the isolation of four new sesquiterpene lactones. Their structures were determined using mass spectrometry, 1-D and 2-D NMR, and (in one case) single crystal X-ray diffraction. All four compounds were cytotoxic in the A2780 human ovarian cancer cell line. Fractionation of an extract of Casimirella ampla led to the isolation of three new diterpenes and two known diterpenes. Their structures were determined using mass spectrometry and 1-D and 2-D NMR. All five compounds were cytotoxic in the A2780 human ovarian cancer cell line. Fractionation of root and stem extracts of Mendoncia cowanii led to the isolation of two new naphthaquinones, and two known naphthaquinones. Their structures were determined using mass spectrometry and 1-D and 2-D NMR. All four compounds were cytotoxic in the A2780 human ovarian cancer cell line and three compounds exhibited weak inhibition of Akt kinase. The fractionation of five additional extracts resulted in the isolation of twelve known compounds. Their structures were determined using mass spectrometry, 1-D and 2-D NMR, and comparison to literature data. All twelve compounds were cytotoxic in the A2780 human ovarian cancer cell line. / Ph. D.

Natural products are biologically active metabolites produced by fungi, bacteria, and plants that have an extended application in pharmaceutical and chemical industries. Because of their chemical versatility, flavoenzymes are commonly involved in natural product biosynthetic pathways. This has given rise to the identification of flavoenzymes that are promising candidates for biomedical and biotechnical applications. This dissertation discusses the characterization of three flavoenzymes involved in natural product biosynthesis. The class B flavin-dependent monooxygenases S-monoooxygenase from Allium sativum (AsFMO) and N-hydroxylating monooxygenase from Streptomyces sp. XY332 (FzmM) were studied. Both enzymes perform heteroatom oxidation as part of allicin or fosfazinomycin biosynthesis respectively. AsFMO was predicted to oxidize S-allyl-L-cysteine (SAC) to alliin in allicin biosynthesis. Surprisingly, AsFMO exhibited negligible activity with SAC, and instead was highly active with allyl mercaptan and NADPH. This contradicted the initial proposal and suggested that AsFMO is involved in an alternative path producing allicin directly from allyl mercaptan. FzmM was identified to perform multiple N-oxidations which lead to the formation of a nitro group. FzmM performed a highly coupled and specific reaction with L-aspartate and NADPH to produce nitrosuccinate. Both AsFMO and FzmM followed a kinetic mechanism representative of class B flavin-dependent monooxygenases with a rapid pro-R stereospecific reduction and the formation of a C(4a)-hydroperoxyflavin intermediate during oxidation. In addition, the AsFMO structure was obtained and consisted of two domains for FAD and NADPH binding signature of class B monooxygenases. The biochemical and structural study of the Acinetobacter baumannii siderophore interacting protein (BauF) was also accomplished. This enzyme is essential in acinetobactin mediated iron assimilation and is important for virulence. The characterization of the binding and reduction of acinetobactin-ferric iron complex revealed that BauF is specific for this substrate and does not utilize NAD(P)H as an electron donor. The unique activity and structure of BauF can aid future drug design. / Doctor of Philosophy / Plants, fungi, and bacteria synthesize and excrete unique chemicals called secondary metabolites or natural products. These compounds are used for many applications including dyes, flavorings, fragrances, and medicine. To make natural products, organisms use enzymes to perform complex reactions. Studying the enzymes that are involved in natural product pathways is important for understanding how secondary metabolites are made. Additionally, these enzymes can be engineered to perform reactions relevant to biotechnical applications. Our lab specializes in the study of flavoenzymes which use flavin chemistry for catalysis. Flavin is a yellow coenzyme that contributes to wide array of reactions by performing 1 or 2 electron transfers. This dissertation discussed the characterization of three flavoenzymes. The first enzyme is a S- monooxygenase from Allium sativum (garlic) called AsFMO. Reported here is the kinetic and structural characterization of AsFMO. We demonstrated that AsFMO was cabable of performing an unexpected reaction with allyl mercaptan likely converting it into allicin, the main flavor ingredient of garlic. Secondly, we reported the kinetic characterization of a nitro- forming enzyme termed FzmM. Nitro- formation is a valuable process as nitro- compounds are used in industrial organic synthesis. It was shown that FzmM performs nitro- formation with high efficiency and is specific for the substrate L-aspartate. Lastly, this work described the characterization of the the siderophore-interacting protein from Acinetobacter baumannii, BauF, which was predicted to be involved in iron acqusition. A. baumannii is a serious human pathogen with multidrug resistance, and inhibiting iron acquisition has been shown to prevent its survival. The characterization of the enzymes involved in this pathway is essential for developing new treatments for A. baumannii infection. We report the structure and function of BauF confirming its role in A. baumannii iron uptake and providing information that will aid in future drug design.

Through our continuing search for anticancer agents from Madagascar rainforests as a part of International Cooperative Biodiversity Group (ICBG), we received two extracts which were active against the A2780 human ovarian cancer cell line and hence were selected for further fractionation. Six compounds were isolated from these extracts. The structure elucidation and characterization of these compounds was carried out using mass spectrometry and 1D and 2D NMR techniques. The bioassay-guided fractionation of Roupellina (Strophanthus) boivinii yielded three new and one known cardenolide glycosides. The structure of the known cardenolide glycoside was determined after comparison of NMR data to that found in literature for digitoxigenin 3-O-β-D-glucopyranosyl-(1â 4)-α-L-acofriopyranoside. All four compounds exhibited good antiproliferative activity on the A2780 bioassay. The fractionation of the extract of Grewia sp. led to the isolation of one new and one known triterpenoid. The known triterpenoid was identified as 7β-hydroxy-23-deoxojessic acid and its structure was confirmed by comparison of its 1D and 2D NMR data to that found in literature. / Master of Science

As a part of the continuing search for bioactive compounds with the Madagascar International Cooperative Biodiversity Group (ICBG), and in collaboration with the Natural Products Discovery Institute of the Institute for Hepatitis and Virus Research (IHVR), thirteen plant extracts were investigated for antiplasmodial activity, thirteen plant extracts were investigated for antiproliferative activity, and one extract was investigated for inhibitors of the shikimate pathway in Plasmodium falciparum. Bioassay-guided fractionation of the extracts led to the identification of nineteen compounds with both antiplasmodial and antiproliferative activity, and thirteen compounds with only antiproliferative activity. Thirteen of these compounds (2.1 – 2.9, 3.3, 3.4, 4.5, and 5.1) were previously unknown. In addition total synthesis was used to confirm the structure of one new compound (4.5) and two other new natural-product like compounds (4.6 and 4.7) were also synthesized and investigated for antiplasmodial activity. / Ph. D.

In our continuing search for biologically active natural products from Madagascar as part of an International Cooperative Biodiversity Group (ICBG) program, a total of four antiproliferative extracts were studied, leading to the isolation of twelve novel compounds with antiproliferative activity against the A2780 human ovarian cancer line, and one extract with antimalarial activities was studied, which led to the isolation of five new natural products with antimalarial activities against the Dd2 and HB3 malarial parasites. The plants and their metabolites are discussed in the following order: one new xanthone and two known guttiferones from Symphonia tanalensis Jum. & H. Perrier (Clusiaceae); four new diphenyl propanes and one new cyclohepta-dibenzofuran skeleton from Bussea sakalava (Fabaceae); four new cardiac glycosides from Leptadenia madagascariensis Decne. (Apocynaceae); two new and four known alkaloids from Ambavia gerrardii (Baill.) Le Thomas (Annonaceae); five new sesquiterpene lactones from Polycline proteiformis Humbert (Asteraceae). The structures of all compounds were determined by analysis of their mass spectrometric, 1D and 2D NMR, UV and IR spectroscopic and optical rotation data. Other than structure elucidation, this dissertation also involve bioactivity evaluation of all the isolates, synthesis of two interesting alkaloids, as well as a proposal for the possible biosynthetic pathway of the new cyclohepta-dibenzofuran skeleton. / Ph. D.