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Thesis advisor: Evan R. Kantrowitz / Thesis advisor: Mary F. Roberts / Living organisms depend on enzymes for the synthesis using small molecule precursors of cellular building blocks. For example, the amino acid aspartate is synthesized in one step by the amination of oxaloacetate, an intermediate compound produced in the citric acid cycle, exclusively by means of an aminotransferase enzyme. Therefore, function of this aminotransferase is critical to produce the amino acid. In the Kantrowitz Lab, we seek to understand the molecular rational for the function of enzymes that control rates for the biosynthesis of cellular building blocks. If one imagines the above aspartate-synthesis example as a single running conveyer belt, any oxaloacetate that finds its way onto that belt will be chemically transformed to give aspartate. We can extend this notion of a conveyer belt to any enzyme. Therefore, the rate at which the belt moves dictates the rate of synthesis. Now imagine many, many conveyer belts lined in a row to give analogy to a biosynthesis pathway requiring more than one enzyme for complete chemical synthesis. This is such the case for the biosynthesis of nucleotides and glucose. Nature has developed clever tricks to exquisitely control the rate of product output but means of altering the rate of one or some of the belts in the line of many, without affecting the rate of others. This type of biosynthetic rate regulation is termed allostery. Studies described in this dissertation will address questions of allosteric processes and the chemistry performed by two entirely different enzymes and biosynthetic pathways. The first enzyme of interest is fructose-1,6-bisphosphatase (FBPase) and its role in the biosynthesis of glucose. Following FBPase introduction in Chapter One, Chapter Two describes the minimal atomic scaffold necessary in a new class of allosteric type 2 diabetes drug molecules to effect catalytic inhibition of <italic>Homo sapiens</italic> FBPase. Following, is the second enzyme of interest, aspartate transcarbamoylase (ATCase) and its role in the biosynthesis of pyrimidine nucleotides. Succeeding ATCase introduction in Chapter Three, Chapter Four describes a body of work exclusively about the catalysis by ATCase. This work was inspired by the human form of the enzyme following the human genome project completion providing data that show likely <italic>Homo sapiens</italic> ATCase is not allosterically regulated. Chapter Five describes work on a allosterically-regulated, mutant ATCase and provides a biochemical model for the molecular rational for the catalytic inhibition upon cytidine triphosphate (CTP) binding to the allosteric site. The experimental techniques used for answering research questions were enzyme X-ray crystallography, <italic>in silico</italic> docking, kinetic assay experiments, genetic sub-cloning and genetic mutation. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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Flavonoids are a specialized group of compounds produced by plants that give them greater adaptability to their environment and ultimately enhance their ability to survive. In plants, one function of flavonoids is to attract pollinators by their various flavor and scent profiles. They also protect the photosynthetic machinery from photo-oxidation. In humans, flavonoids have been shown to act as antioxidants, exhibit antimicrobial activity, and have shown potential as cancer treatments. In nature, flavonoids are most often found coupled with a sugar group (glucose, rhamnose, and others) which imparts stability and increases bioactivity. The process of adding a sugar (glycosylation) is catalyzed by a class of enzymes called glycosyltransferases (GT). One such enzyme found in grapefruit only glucosylates the flavonol class of flavonoids at the 3-OH position and is of interest due to its unique substrate and regio-specificity. Called Cp3GT (Citrus paradisi flavonol 3-O glucosyltransferase), this enzyme is similar in structure to other plant GT’s yet differs in the flavonoids it can glucosylate and where the glucose can be added. To date, the literature has not reported a structural mechanism for a flavonol specific 3-O glucosyltransferase’s unique catalytic activity. High-resolution structural imagery of enzymes, elucidated using X-ray crystallography, can be used to direct custom enzyme development to produce bioavailable natural products. Furthermore, structural research on enzymes with high specificity strengthens enzyme-ligand docking simulations, which are commonly used to test the binding affinity of potential pharmaceuticals. This research hypothesizes Cp3GT has structural features that confer its unique substrate and regiospecificity that are not revealed by homology modeling. This hypothesis will be tested using x-ray crystallography of purified Cp3GT protein bound to its preferred flavonol substrates. The gene for Cp3GT was transformed into Pichia pastoris and was recombinantly expressed using methanol induction. Cp3GT was purified to 80% purity using cobalt metal affinity chromatography. Cp3GT was subjected to additional purification measures using anion exchange chromatography with the goal of increasing purity to ≥95% for crystallization experiments. Purity analysis was conducted using SDS-PAGE (Coomassie/silver stain, western blot) and UV-Vis spectrophotometry. While initial results are promising, additional purification steps may be needed to achieve the purity necessary for crystallization.

Dynamic kinetic resolution (DKR) is a powerful method for obtaining compounds with high optical purity. The process relies on the combination of a kinetic resolution with an in situ racemization. In this thesis, a combination of an immobilized hydrolase and a transition metal-based racemization catalyst was employed in DKR to transform racemic alcohols and amines into enantioenriched esters and amides, respectively. In the first part the DKR of 1,2-amino alcohols with different rings sizes and N-protecting groups is described. We showed that the immobilization method used to support the lipase strongly influenced the stereoselectivity of the reaction. The second part deals with the DKR of C3-functionalized cyclic allylic alcohols affording the corresponding allylic esters in high yields and high ee’s. The protocol was also extended to include carbohydrate derivatives, leading to inversion of a hydroxyl substituted chiral center on the carbohydrate. The third part focuses on an improved method for obtaining benzylic primary amines. By using a novel, recyclable catalyst composed of Pd nanoparticles on amino-functionalized mesocellular foam, DKR could be performed at 50 °C. Moreover, Lipase PS was for the first time employed in the DKR of amines. In the fourth part DKR was applied in the total synthesis of Duloxetine, a compound used in the treatment of major depressive disorder. By performing a six-step synthesis, utilizing DKR in the enantiodetermining step, Duloxetine could be isolated in an overall yield of 37% and an ee &gt;96%. In the final part we investigated how the enantioselectivty of reactions catalyzed by Candida Antarctica lipase B for δ-substituted alkan-2-ols are influenced by water. The results showed that the enzyme displays much higher enantioselectivity in water than in anhydrous toluene. The effect was rationalized by the creation of a water mediated hydrogen bond in the active site that helps the enzyme form enantiodiscriminating binding modes. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

<p>The quantum chemical investigations presented in this thesis use hybrid density functional theory to shed light on the catalytic mechanisms of mononuclear non-heme iron oxygenases, accommodating a ferrous ion in their active sites. More specifically, the dioxygen activation process and the subsequent oxidative reactions in the following enzymes were studied: tetrahydrobiopterin-dependent hydroxylases, naphthalene 1,2-dioxygenase and α-ketoglutarate-dependent enzymes. In light of many experimental efforts devoted to the functional mimics of non-heme iron oxygenases, the reactivity of functional analogues was also examined.</p><p>The computed energetics and the available experimental data served to assess the feasibility of the reaction mechanisms investigated. Dioxygen activation in tetrahydrobiopterin- and α-ketoglutarate-dependent enzymes were found to involve a high-valent iron-oxo species, which was then capable of substrate hydroxylation. In the case of naphthalene 1,2-dioxygenase, the reactivity of an iron(III)-hydroxperoxo species toward the substrate was investigated and compared to the biomimetic counterpart.</p>

[NiFe] hydrogenases are microbial metalloenzymes that catalyse the reversible interconversion between molecular hydrogen and protons with high selectivity and efficiency. The catalytic properties of different [NiFe] hydrogenases vary according to the physiological roles they each play, yet all seem to be based upon an almost identical catalytic site architecture. Through efforts to understand the structural and mechanistic basis for the differing properties of [NiFe] hydrogenases, it has become increasingly evident that electron transfer to and from the active site, mediated by a set of Iron-Sulphur clusters, influences to a significant extent the observed catalytic properties of different hydrogenases. Here we present a comprehensive study of E. coli Hyd-1, an O₂-tolerant hydrogenase, by PFE with a focus on the properties that are characteristic of O₂-tolerant enzymes: overpotential requirement, lack of H₂ production, low K^H₂<sub style='position: relative; left: -1.2em;'>M</sub>, and high E_switch. We show that Hyd-1 catalysis can be made reversible by increasing the equilibrium potential for the reaction through changes in substrate concentration, and that electron transfer into and out of the enzyme molecule, rather than active site properties, is responsible for the characteristics of overpotential and bias in Hyd-1. We present a set of experiments with Hyd-2 from E. coli in which surface-exposed cysteine residues are specifically introduced near the distal and medial Iron-Sulphur clusters to act as points of attachment for photosensitizer molecules, and a study of the kinetics of electron injection from photoexcited molecules to the enzyme and subsequent absorbance changes attributed to transient redox changes at the active site. We are able to show lightdependent H2 production from a Hyd-2 + photosensitizer system. Finally, we present the first purification of the formate-hydrogen lyase (FHL) complex from E. coli, the complex responsible for H2-production by this organism during fermentation, and we provide a characterisation of the complex by EPR and PFE. The properties of Hyd-3, the hydrogenase subunit of the FHL, seem to differ from those observed previously for other [NiFe] hydrogenases.

The study herein described involved the detection of pyridine N-oxide reductase activity in cell-free extracts of E. coli 9723, the determination of co-factors necessary for the enzymatic process, a study of the optimum conditions for enzyme catalysis, and a general characterization of the enzyme.