Katherine Ryan
Relevant Thesis-Based Degree Programs
Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
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Natural products are essential to the discovery of new drugs, including antibiotics. Industrial interest in natural products has declined since the 1980s, but advances in biocatalysis and biosynthetic knowledge have helped revive interest in natural products as these advances contribute to more feasible discovery, production and derivatization of natural products. To continue this industrial interest in natural products and their related compounds, work should be done to accumulate more biosynthetic knowledge to further improve methods of discovery, production and derivatization and facilitate more widespread use of biocatalysts. Indolmycin is a natural product with antibiotic activities against methicillin-resistant Staphylococcus aureus, Helicobacter pylori and Plasmodium falciparum, whose biosynthetic pathway is shown here to be a source of new biochemical tools. First, in order to better understand the unique reactivity of the rare oxygen- and pyridoxal 5′-phosphate (PLP)-dependent arginine desaturases discovered from the indolmycin biosynthetic pathway, the first X-ray crystal structure of an arginine desaturase was solved. This structure showed an active site that was highly similar to the related oxygen- and PLP-dependent hydroxylases. Catalytic residues for the arginine desaturases were uncovered by creating mutagenic variants based on the crystal structure information. Second, sequence similarity analysis and side-product analysis were done, which further supported a higher similarity to the arginine hydroxylases than was originally predicted. Additionally, superoxide was shown to be an intermediate of the arginine oxidase mechanism for the first time through EPR and cytochrome c assays. Based on this information, a unified mechanistic hypothesis is proposed which suggests that desaturation and hydroxylation may be differentiated by the presence/position of water in the active site.Third, the indolmycin biosynthetic enzymes are used in conjunction with a promiscuous tryptophan synthase and a three-step chemical synthesis to produce indolmycin and several novel halogenated derivatives. Derivatives with fluorinated indole substitutions showed a moderate bioactivity against S. aureus and could be useful in developing indolmycin for clinical use. Overall, this work uses the indolmycin biosynthetic enzymes to expand the known biocatalytic repertoire with the hope that it can contribute to more widespread use of biocatalysts in the production of natural product-derived molecules.
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The discovery of novel natural products continues to be critical for the development of new pharmaceuticals. Innovative methods to discover novel natural products can reveal previously overlooked chemical diversity. One such method is genome mining, where sequenced bacterial genomes are assessed for the presence of biosynthetic gene clusters. Another such method is bioassay-guided fractionation. Following either of these approaches, the bacteria must be grown and harvested, and novel natural products must be isolated and characterized. In the first part of this thesis, a nitrogen-NMR guided approach was developed to retrieve genetically predicted natural products from bacterial cultures. Piperazic acid (Piz)-containing natural products were targeted because this unique amino acid is often found in peptidic natural products with biological activity and impressive chemical structures. Piz contains a unique nitrogen-proton NMR correlation targeted through ¹H-¹⁵N HSQC. The unique N-H correlation also gave access to Piz’s diagnostic spin system through ¹H-¹⁵N HSQC-TOCSY NMR experiments. These two ¹⁵N NMR experiments were used to monitor for the presence of peptides containing Piz in culture extracts of genome-mined bacteria. Through the application of these ¹⁵N NMR experiments to guide isolation of Piz natural products, four novel compounds were discovered from Streptomyces incarnatus NRRL 8089. Three of these were isolated and structure’s elucidated as part of this thesis work: dentigerumycin F (4.2), dentigerumycin G (4.1) and incarnatapeptin A (4.3). 4.3 demonstrated a unique bicyclic moiety not previously seen in chemical structures, and a fourth compound, incarnatapeptin B (4.4), has in vitro cytotoxicity. In the second part of the thesis, bioassay-guided fractionation is used to screen a small library of marine bacteria in various assays. Using this method, known natural product molecules were uncovered, along with the discovery of two novel natural products from the marine bacterium Salinispora arenicola RJA3005. These two compounds, 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-hydroxy-3-methyl-2H-pyran-2-one (6.1) and N-(3-hydroxy-5-(1-hydroxy-2-(4-hydroxy-3-methyl-2-oxo-2H-pyran-6-yl)propyl)phenyl)acetamide (6.4), were isolated from extracts of wild-type bacteria for the first time. Feeding studies and analysis of ¹³C splitting patterns suggest that these compounds were biosynthesized from bacterium through phosphoenolpyruvate and erythrose precursors. Altogether, this thesis's work develops and harnesses various natural product discovery methods to uncover diverse natural products.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
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N-nitroso containing natural products, which contain the core structure −N−N=O, can act as both chemotherapeutics and as carcinogens due to their ability to alkylate DNA. While the enzymes responsible for the formation of this functional group have also been proposed as potential green alternatives to traditional synthetic methods, little is known about their mechanisms of action. One N-nitroso containing natural product, streptozocin, has been used as a chemotherapeutic since the 1960s, but the biosynthetic machinery responsible for its synthesis remained unknown until recent years.In this research, I obtained crystal structures of two enzymes involved in the biosynthesis of streptozocin: StzF, the enzyme that is believed to form the N-N bond, and StzK, an enzyme with a yet-unknown function that is proposed to ligate an N-nitroso-containing-compound to an unidentified glucosamine donor. The StzF and StzK structures were obtained at resolutions of 1.6 and 1.8 Å, respectively. StzF is a unique enzyme composed of 3 domains: an N-terminal domain, a diiron domain, and a cupin domain. An anomalous signal was observed that indicates that StzF binds iron. To explore the behavior of the metal centers of this enzyme, EPR studies were conducted that allowed for the detection of an iron-nitrosyl species that may be an intermediate in N-N bond formation. To probe the prevalence of N-nitroso compounds in the environment, bioinformatic searches were conducted. The presence of sequences encoding N-nitroso forming machinery was observed in a variety of deposited genomes, suggesting that these compounds may be isolable from the corresponding organisms.
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Chiral amine moieties are widely distributed in bioactive natural products and pharmaceutical ingredients. NAD(P)H-dependent imine reductases (IREDs) have been identified as potential biocatalysts for chiral amine synthesis via asymmetric reduction of the imine substrates. In this work, I characterized two unusual imino-acid-reducing enzymes, Punc5 and Bsp5, from the D-2-hydroxy-acid dehydrogenase (DHDH) family. The DHDH enzymes are known for reducing α-keto acids directly to the corresponding chiral hydroxy acids; however, both Punc5 and Bsp5 demonstrate imine reductase activity. Specifically, when coupled with L-arginine oxidase Ind4, both enzymes can use the coenzyme NAD(P)H to stereo-specifically reduce the Ind4 products didedydroarginine and dedydroarginine to D-4,5-dehydroarginine and D-arginine, respectively. Furthermore, Punc5 shows a DHDH activity, converting 2-ketoarginine to 5-guanidino-2-hydroxypentanoic acid. Both IREDs and DHDHs belong to the NAD(P)H-dependent oxidoreductase family; however, imine reduction catalyzed by DHDHs had never been reported before. To understand how Punc5 and Bsp5 evolved from DHDHs with asymmetric imino-acid-reducing activities, and to offer insights into NAD(P)H-dependent oxidoreductases’ chemoselectivity, I obtained ~1.6 Å resolution ternary structures of each enzyme bound with coenzyme NADPH and product D-arginine. These ternary structures of Punc5 and Bsp5 at high resolution closely resemble typical DHDHs; however, the spatial relationship of the coenzyme, product, and catalytic residues within the active site suggests a different catalytic mechanism from typical DHDHs. Structure-guided mutagenesis work uncovered an essential residue Tyr97 for substrate binding in Punc5. Biochemical characterization of the Punc5-Y97F variant suggests imine reduction under the acidic condition is a more facile reaction compared to ketone reduction as Punc5-Y97F is active towards imino acids, but it is inactive towards 2-ketoarginine.This unique imino-acid-reducing activity demonstrated by Punc5 and Bsp5 indicate that other subfamilies of NAD(P)H-dependent oxidoreductases besides known IREDs could also have the potential to produce chiral amines and be applied in pharmaceutical industry. Besides, our work offered three-dimensional frameworks for understanding how these unusual imino acid reductases differ from typical DHDHs, setting the stage for further engineering efforts to either enhance their catalytic efficiency or expand their substrate scopes.
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Cladoniamides are a set of bisindole compounds that contain an indolotryptoline rather than the more common indolocarbazole scaffold. Besides their interesting structures, several of the cladoniamides have been found to be potent cytotoxic agents. We set out to isolate the brominated analogues of known cladoniamides by supplementing the fermentation medium with KBr, which led to the production of 5-bromocladoniamide A. However, the observed production levels were very low. To determine whether the selection against the bromo-substrates is early or late in the cladoniamide biosynthetic pathway, we synthesized 3-chloroarcyriaflavin and 3-bromoarcyriaflavin. These substrates were then fed into Streptomyces albus + cla (ΔclaC), which contains the complete cladoniamide biosynthetic pathway, except one crucial gene required for the production of cladoniamides. Through the feeding experiment, we found approximately equal amount of incorporation of the chloro and bromo substrates. The results suggest that the substrate selectivity against bromo precursors is upstream in the pathway from the enzyme encoded by the inactivated gene. Overall, we have observed the production of brominated cladoniamides through the two different methods of modifying the growth conditions and of precursor directed biosynthesis. Furthermore, this work presents a facile way to generate new indolotryptoline molecules through synthetic generation of desired indolocarbazole substrates and then biological conversion using the cladoniamide biosynthetic pathway.
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