Katherine Ryan

Natural products continue to serve as a rich source of bioactive molecules and interesting chemistry. However, contemporary endeavours are often frustrated by the re-isolation of known compounds. A variety of approaches emerged for targeting novel natural products for isolation. Rapid advancements in genomics have enabled ‘genome-mining’ techniques, frequently deployed when studying microbes, using bioinformatics-based predictions of genes encoding biosynthetic enzymes. This work describes the application of a hybrid ‘genome-mining’ and spectroscopic screening approach leading to isolation of the biologically potent and chemically novel caveamides. The dynamic structures of caveamides presented a challenging structure elucidation, requiring detailed interpretations of spectroscopic data and chemical degradation experiments.Chapter 1 gives a brief overview of the field, and explains the hybrid genome-mining/spectroscopy approach previously described by our groups. The method targets the isolation of compounds containing piperazate residues, cyclic hydrazine amino acids that frequently coincide with bioactivity and structural novelty. Genes encoding piperazate biosynthesis can be identified, and piperazate residues give a distinct NMR spectroscopic signature.Chapters 2 and 3 describe piperazate-targeting isolation of caveamides A (1) and B (2) from Streptomyces sp. strain BE230, a soil microbe collected from New Rankin Cave (Missouri). These chapters describe detailed structure elucidation, as well as the results of in vitro bioassays. In addition to piperazate residues, caveamides exhibit a rare cyclohexenylalanine moiety, not previously seen as a free peptide residue. This residue is hydrolytically unstable, and assigning its configuration necessitated a novel use of dibromination to preserve stereochemical information, as described in Chapter 3.Chapter 4 describes the identification of caveamide congeners by taking advantage of diagnostic mass spectrometry fragmentation. It also explores the generalizability of this screen using public data, setting the stage for further research.Chapter 5 describes the biogenetic considerations of caveamides, placing their structures in the context of known biosynthetic pathways. Notably, the isolation of caveamides along with their putative biosynthetic gene cluster enables some inferences about cyclohexenylalanine biosynthesis, which is poorly understood. This chapter provides an interpretation of previously reported experimental findings in light of new insights afforded by caveamides. Chapter 6 concludes by proposing future avenues for investigation.

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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 a chemically diverse group of molecules that are isolated from bacteria, fungi, or plants. Many natural products can be employed as active pharmaceutical agents, among which there is a subset bearing N-heterocycles or diazo groups in their structures. In this thesis, I have been studying the biosynthetic assembly of two types of heterocycle-containing molecules and one type of diazo compound. In the first chapter of my thesis, I review the research background of these three molecules: piperazic acid, azaserine, and azomycin. In the second chapter of my thesis, I describe my work on piperazic acid, which for the first time reveals how this cyclic hydrazine α-amino acid is activated for incorporation into diverse nonribosomal peptidyl natural products by employing a strain named Streptomyces incarnatus NRRL 8089 and its putative incarnatapeptin gene cluster. In the third chapter of my thesis, I discuss the biosynthetic studies of the diazo α-amino acid azaserine in Glycomyces harbinensis DSM46494, implicating a novel pathway for diazo group formation in nature. In the fourth chapter of my thesis, I write about my X-ray crystallographic studies on two azomycin biosynthetic enzymes from the Pseudomonas genus − RohQ and RohS − featuring a cyclodehydratase catalyzing spontaneous cyclodehydration reaction and an enzyme belonging to the emerging family of heme oxygenase-like ḏiiron oxidase/oxygenase (HDO) that catalyzes a six-electron oxidation, respectively. In the concluding chapter of my thesis, I summarize my work on these three research topics and discuss their future directions.

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Studying individual biosynthetic transformations in the creation of natural products often reveals surprising and powerful chemical reactivities and molecular handling strategies. Indolocarbazole bisindoles have been widely tested in clinical studies and arise from oxidative dimerization of L-tryptophan. Among bisindoles, reductasporine bears an unusual dimethylpyrrolinium structure. Its biosynthesis differs from other indolocarbazole pathways by two tailoring enzymes: the imine reductase RedE and N,N-dimethyltransferase RedM. Here I reconstitute this pathway in vitro and show that RedE prevents unstable indolocarbazole intermediates from becoming oxidized and provides reduced didemethylreductasporine substrate to RedM. Employing X-ray crystallography, I solved two ternary complexes of RedE co-crystallized with the substrate-mimic arcyriaflavin A, revealing an extended active site cleft with distinct secondary indolocarbazole binding site. Site-directed mutagenesis confirms the conserved active site aspartate (D168) is essential for activity and anchors the substrate via hydrogen-bonding. Variants targeting the secondary binding site reduce catalytic efficiency, suggesting this site protects the substrate from autooxidation. I solved the 1.7 Å structure of RedM demonstrating it adopts distinct open and closed conformations with either SAH or SAM cofactor, respectively. Site-directed mutagenesis, docking and sequence bioinformatics identify conserved substrate-recognizing residues and suggest dimethyltransferase catalytic activity likely arises from precise orientation and desolvation of the substrate. Recently, Cap15 has been shown to be an oxygen- and pyridoxal phosphate (PLP)-dependent enzyme and is the first example of this activity in the L-seryl-tRNA(Sec) selenium transferase enzyme family. It catalyzes oxidative-decarboxylation of 5ʹ-glycyl uridine to the corresponding 5ʹ-carboxamide uridine. Solving for the 2.40 Å resolution crystal structure of Cap15 shows PLP bonds to K230 and a phosphate anion in the active site bridges N- and C-terminal domains. Sequence analysis reveals the loop proximal to the internal aldimine and hydrogen bonding to the active site phosphate is strictly conserved among Cap15 homologues present in capuramycin-type gene clusters. The crystal structures provide a basis for further investigations into the sequence-determinants of secondary binding site formation in RedE, dimethylation activity in RedM and oxygen-consuming activity in Cap15. RedE in particular can serve as a starting-point in the engineering of imine reductases to accommodate large substrates in the production of industry-relevant chiral amines.

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Over time, evolution can result in enzymes developing novel functions. An emerging example of this phenomenon comes from studies of pyridoxal-5ʹ-phosphate (PLP)-dependent enzymes, which catalyze a diverse set of chemical reactions on amino acid substrates. This thesis describes the discovery and characterization of some unusual PLP-dependent enzymes.Some PLP-dependent enzymes have been shown to catalyze challenging oxidations of an L-arginine substrate using O₂ as a co-substrate. In Chapter 2 I set out to describe new PLP-dependent arginine oxidases and study how they function. Using bioinformatics, I was able to hone in on one particular enzyme, named RohP. Biochemical characterization of RohP revealed that it is an arginine hydroxylase, which catalyzes the formation of (S)-4-hydroxy-2-ketoarginine. Furthermore, I was able to obtain several high-resolution X-ray crystal structures of RohP at different stages of its catalytic cycle. Together these results advance the understanding of how O₂- and PLP-dependent enzymes function.RohP was found in a conserved five gene biosynthetic gene cluster, with no known product. Therefore, in Chapter 3 I set out to determine what the product of this unusual biosynthetic gene cluster was. Using the studies of RohP as a starting point, additional in vitro biochemical investigations of four other enzymes encoded along with RohP in this biosynthetic gene cluster revealed that together they convert L-arginine to the antibiotic azomycin (2-nitroimidazole). As azomycin was first isolated over 50 years ago, the discoveries described in this chapter solve a longstanding biosynthetic mystery. Interesting PLP-dependent enzymes are found in many biosynthetic pathways. In Chapter 4 I report my characterization of BesB, an unrelated PLP-dependent enzyme which catalyzes the formation of a terminal alkyne bond. BesB has limited solubility in E. coli, which has hampered its study initially. Through use of a different heterologous expression system I was able to obtain soluble BesB. Through biochemical and X-ray crystallographic analysis, an active site phenylalanine substitution appears to be key to unlocking the novel reactivity of BesB. This study provides the first crystal structures of any alkyne-forming enzyme. Insights from the studies of RohP and BesB should prove useful in developing novel PLP-dependent biocatalysts.

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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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