The amidohydrolase superfamily is a functionally diverse group of evolutionarily related proteins which utilize metal cofactors in the activation of a hydrolytic water molecule and in the stabilization of the resulting tetrahedral intermediate. Members of this superfamily have been described which use one or two divalent transition metals. These metal cofactors are located in either or both of two active-site metal binding centers which are labeled as the Ma and MB sites. The goal of this research was to elucidate the nature of the reactions catalyzed by Ma and MB mononuclear members of the amidohydrolase superfamily. This was approached through comprehensive mechanistic evaluations of two enzymes which utilized the different metal sites. Nacetyl- D-glucosamine-6-phosphate deacetylase from E. coli (NagA) and cytosine deaminase from E. coli (CDA) served as models for mononuclear amidohydrolase superfamily enzymes which have evolved to utilize a single B-metal and a single a-metal for hydrolysis, respectively. This research elucidated the different properties imparted by the distinct a and B active sites and the specific interactions utilized by the enzymes for substrate binding and catalysis. These studies led to the eventual proposal of detailed chemical mechanisms and the identification of rate determining steps. Knowledge of sequence-function relationships was applied toward the discovery of function for enzymes related to cytosine deaminase and guanine deaminase. The first group of enzymes investigated was proposed to catalyze the fourth step in riboflavin and coenzyme F420 biosynthesis in Achaea. Three putative deaminases; Mm0823 from Methanosarcina mazei, MmarC7_0625 from Methanococcus maripaludis C7 and Sso0398 from Sulfolobus solfataricus were cloned and expressed. These proteins proved to be intractably insoluble. A second set of enzymes, Pa0142 from Pseudomonas aeruginosa PA01 and SGX-9236e (with crystal structure PDB: 3HPA) were found to catalyze the novel deamination of 8-oxoguanine, a mutagenic product of DNA oxidation. 9236e was cloned from an unidentified environmental sample of the Sargasso Sea. The closest homolog (98% identical) is Bcep18194_A5267 from Burkholderia sp. 383. Additionally, it was discovered that the proteins SGX-9339a (with crystal structure PDB: 2PAJ) and SGX-9236b catalyzed the deamination of isoxanthopterin and pterin-6- carboxylate in a poorly characterized folate degradation pathway. These enzymes were also from unknown environmental samples of the Sargasso Sea. The closest homolog of 9339a (88% identical) is Bxe_A2016 from Burkholderia xenovorans LB400. The closest homolog of 9236b (95% identical) is Bphyt_7136 from Burkholderia phytofirmans PsJN.

Diverse biological activities of polyketide natural products are often associated with specific structural motifs, biosynthetically introduced after construction of the polyketide core. Therefore, investigation of such "post-polykektide synthase (PKS)" modifications is important, and the accumulated knowledge on these processes can be applied for combinatorial biosynthesis to generate new polyketide derivatives with enhanced biological activities. In addition to the practical value, a lot of unprecedented chemical mechanisms can be found in the enzymes involved therein, which will significantly advance our understanding of enzyme catalysis. The works described in this dissertation focus on elucidating a number of post-PKS modifications involved in the biosynthesis of an insecticidal polyketide, spinosyn A, in Saccharopolyspora spinosa. First, three methyltransferases, SpnH, SpnI, and SpnK, responsible for the modification of the rhamnose moiety, have been investigated to verify their functions and to study how they are coordinated to achieve the desired level of methylation of rhamnose. In vitro assays using purified enzymes not only established that SpnH, SpnI, and SpnK are the respective rhamnose 4ʹ-, 2ʹ-, and 3ʹ-O-methyltransferase, but also validated their roles in the permethylation process of spinosyn A. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK, and SpnH in sequence is productive for the permethylation of the rhamnose moiety, which is likely achieved by the proper control of the expression levels of the methyltransferase genes involved in vivo. The key structural feature of spinosyn A is the presence of the unique tetracyclic architecture likely derived from the monocyclic PKS product. To elucidate this "cross-bridging" process, which had been hypothesized to involve four enzymes, SpnF, SpnJ, SpnL, and SpnM, the presumed polyketide substrate was chemically synthesized using Julia-Kocienski olefination, Stille cross-coupling, and Yamaguchi macrolactonization as key reactions. Incubation of the synthesized substrate with SpnJ produced a new product where the 15-OH group of the substrate is oxidized to the ketone. Next, it was demonstrated that incubation of this ketone intermediate with SpnM produces a tricyclic compound, via a transient monocyclic intermediate with high degree of unsaturation. Whereas it was initially thought that SpnM catalyzes both dehydration and [4+2] cycloaddition in sequence, detailed kinetic analysis revealed that SpnM is only responsible for the dehydration step, and the [4+2] cycloaddition step is indeed catalyzed by SpnF. Finally, successful conversion of the tricyclic intermediate to the tetracyclic core was demonstrated using SpnL. Proposed chemical mechanisms of SpnF and SpnL, Diels-Alder and Rauhut-Currier reactions, respectively, are interesting because enzymes capable of catalyzing these reactions have yet to be characterized in vitro. This work not only establishes the biosynthetic pathway for constructing the spinosyn tetracyclic core, but also epitomizes the significance of the post-PKS modification as a rich source of new enzyme catalysis. / text

Flavin-dependent monooxygenases (FMOs) are a large family of enzymes found in microbes, plants, animals, and humans involved in defense pathways, xenobiotic metabolism, and natural product biosynthesis. One class of FMOs, Class B, carries out the oxidation of heteroatomic substrates, via hydroxylation, S-oxygenation, Baeyer-Villiger oxidation, and decarboxylation, using NAD(P)H as a coenzyme. In this dissertation, the characterization of several FMOs from bacteria and plants is described. The putrescine N-monooxygenase (NMO) FbsI from Acinetobacter baumannii is involved in the fimsbactin siderophore biosynthetic pathway, a virulence factor that allows acquisition of free iron from a human host by a pathogen. We show that putrescine is hydroxylated to form N-hydroxyputrescine and is favored over the aliphatic diamine cadaverine and amino acid L-ornithine. The three-dimensional structure of FbsI was solved and shown to have similarities to other NMOs, and characterization of active site mutants revealed residues essential for catalysis and cofactor specificity. The flavin-dependent S-monooxygenase TvMAS1 from the society garlic Tulbaghia violacea has been implicated in the production of marasmin, a natural product with human health benefits. We find that TvMAS1 has a broad substate scope among thiol and sulfide-containing compounds, particularly L-cysteine derivatives. Additionally, we show that S-allyl-L-cysteine is the preferred substrate and propose TvMAS1 to primarily have a physiological role in allicin, not marasmin biosynthesis. Lastly, we characterized the auxin-producing FMO YUC10 from Arabidopsis thaliana and showed the enzyme to only have steady-state activity with aromatic α-keto acids indole-3-pyruvic acid (IPA) and phenylpyruvic acid (PPA). We also propose that a C4a-peroxyflavin intermediate acts as a nucleophile to perform the oxidative decarboxylation on IPA and PPA. The work in this dissertation fills several knowledge gaps among bacterial and plant FMOs and with the established mechanisms aims to guide future drug discovery, green chemistry, and agricultural bioengineering efforts. / Doctor of Philosophy / The water-soluble vitamin riboflavin, also known as Vitamin B2, is an essential nutrient with numerous human health benefits and known for its characteristic yellow-orange color. The major role of riboflavin is in the synthesis of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are involved in biological processes such as energy metabolism and detoxification. Flavoenzymes are proteins found in all living organisms that use either FMN or FAD to facilitate otherwise unfavorable biochemical reactions in living systems. One class of flavoenzymes, known as flavin-dependent monooxygenases (FMOs), carries out oxidation-reduction reactions on an array of small molecules with the aid of molecular oxygen. In this dissertation, we present the characterization of three FMOs and propose their roles in nature. The pathogenic bacterium Acinetobacter baumannii can survive in human hosts using several virulence factors, one of which are siderophores – molecules that scavenge iron and transport it back to the organism. We determined that the enzyme FbsI hydroxylates the amine-containing substrate putrescine into N-hydroxyputrescine, a building block of the A. baumannii siderophore fimsbactin A. By solving the enzyme's three-dimensional structure and characterizing its reaction mechanism, we hope to guide future drug discovery studies of this target. We also describe the role of two FMOs from the society garlic Tulbaghia violacea (TvMAS1) and the thale cress Arabidopsis thaliana (YUC10). We show that TvMAS1 performs S-oxidation of S-allyl-L-cysteine to alliin with high efficiency, implicating a role in the production of allicin - a flavorful compound with health benefits. Finally, we show that YUC10 is involved in the production of auxin, a plant hormone that directs growth and development. A novel chemical mechanism for the YUCCAs is proposed, providing insight from in vitro experiments to guide in vivo findings. Our work with TvMAS1 and YUC10 will help guide future protein engineering and green chemistry efforts in the agricultural industry.

Natural products are secondary metabolites produced by plants and microorganisms that often possess medicinal properties and are implicated in organismal defense. Drawbacks to utilizing natural products in the pharmaceutical industry are difficulties with isolation from biological sources and low yields that can lack stereospecificity from synthetic sources. It is paramount to solve these issues and to develop novel natural products to combat the growing antimicrobial resistance crisis, which was responsible for ~5 million deaths in 2019 alone. One approach is utilizing enzymes to synthesize existing natural products to improve the yields and stereospecificity issue. This dissertation is focused on the biochemical characterization of three enzymes-ZvFMO, OxaD, and CreE-that are implicated in the detoxification of natural products used for organismal defense or participate in the biosynthesis of novel natural products. Each of these enzymes belong to the flavin-dependent monooxygenase (FMO) family, which catalyze the oxygenation of a substrate, generating an oxidized product. ZvFMO, from the insect food crop pest, Zonocerus variegatus, was determined to catalyze a highly uncoupled oxygenation reaction of the nitrogen or sulfur atom of various substrates. OxaD, from Penicillium oxalicum F30, catalyzes novel sequential oxidation reactions of the indole nitrogen of roquefortine C. CreE, from Streptomyces cremeus, also catalyzes sequential nitrogen oxidation reactions to convert L-aspartate to nitrosuccinate en route to biosynthesis of cremeomycin. For each enzyme, the steady-state kinetics have been determined using an oxygen consumption assay and the rapid-reaction kinetics were measured using anaerobic time-resolved spectroscopy. All three enzymes feature a fast flavin reduction step and a slow flavin dehydration step. The oxygenation chemistry of each enzyme was found to proceed through a highly reactive oxygenating species, the C4a-hydroperoxyflavin. Site-directed mutagenesis efforts led to the identification of key active site residues involved in flavin motion and substrate binding, revealing important information about the active site architecture for enzyme engineering applications and drug discovery efforts. / Doctor of Philosophy / Natural products are compounds that are produced by many plants, fungi, and bacteria that have potent medicinal properties and can be used to defend the organism against pests. Unfortunately, using these compounds widely in the pharmaceutical industry is difficult because it is hard to isolate the compound of interest from the organism that produces it and attempts to produce it chemically can result in low yields. Additionally, the overuse of the current natural products, which are most of the antibiotics on the market today, has led to an extreme increase in the resistance of bacteria, fungi, and parasites to the natural product-based drug. Therefore, it is essential that a method is developed to produce novel natural products at high yields to combat the antimicrobial resistance crisis. One method is by using enzymes to generate the natural products of interest. Enzymes are biological catalysts that speed up reactions by ensuring that less energy is required to transition from a reactant to a product and are highly efficient. This dissertation focuses on the characterization of three enzymes that could aid in our understanding of natural product chemistry. All three enzymes insert an oxygen atom on a nitrogen of their respective reactant. The first enzyme ZvFMO, is from an insect and its reactivity causes the insect to become resistant to the natural product-based plant defense mechanism, demonstrating that ZvFMO is a great candidate for inhibitor design. OxaD is the second enzyme and is involved in producing natural products that have antimicrobial and anticancer properties. The last enzyme, CreE, is involved in generating the natural product, cremeomycin, which possesses potent antimicrobial and anticancer properties as well. The reactions of OxaD and CreE positions these enzymes as candidates to produce novel natural products and other efforts to expand their reactivity. The rates of each reaction step have been determined in this work. Key amino acids that contribute to the reaction chemistry and the uptake of the reactant have been identified, laying a solid foundation for drug discovery efforts.

The first identfied 2-oxoglutarate (2OG) dependent oxygenase was a collagen modifying enzyme in the work by Hutton et al. in 1967. Subsequent work has revealed that 2OG dependent oxygenases are a large family with diverse biological roles. With small molecule substrates, these enzymes catalyse a wide range of oxidative reactions, including those that form part of antibiotic biosynthetic pathways. The currently accepted consensus mechanism for catalysis by 2OG-dependent oxygenases is based on crystallographic data, kinetics and on quantum chemical calculations. The consensus mechanism involves oxidative decarboxylation of 2OG by reaction with an oxygen molecule producing CO₂, succinate and a reactive oxidising species that reacts with the 'prime' substrate. Deacetoxycephalosporin C synthase (DAOCS) is a 2OG-dependent oxygenase involved in cephalosporin biosynthesis. The mechanism of DAOCS is of particular interest because it has recently been proposed to be different from the consensus mechanism. The new mechanism proposal from Valeg ard et al. is primarily based on high-resolution crystallographic data with support from steady-state kinetic experiments and quantum-chemical calculations. The work in discussed in this thesis aimed to test the proposal of Valegård et al. by using a combination of spectroscopic and spectrometric methods analysing enzyme-substrate interactions. Substrate binding was investigated using both protein-observe (Chapter 3) and ligand-observe (Chapter 4.1 and 4.2) methods. Preliminary UV-visible data on enzyme-substrates complex formation was also obtained. The strength of substrate and cosubstrate binding was characterised through dissociation constant measurement. An activity assay (Chapter 2) that allows for direct and simultaneous monitoring of 2OG decarboxylation and penicillin ring expansion was optimised. Both the ligand-observe and protein-observe binding experiments as well as the preliminary UV-visible data indicate that the formation of a ternary complex between DAOCS, 2OG and the penicillin substrate is viable. The activity assay conclusively showed that in the presence of unnatural substrates, such as penicillin G, 2OG oxidation is significantly uncoupled from penicillin oxidation. Uncoupled turnover does not occur in the presence of the natural substrate, penicillin N, which is an aspect that should be considered in the analysis of the steady-state kinetic data. Overall, the results provide evidence that, the consensus mechanism for 2OG-dependent oxygenases is viable for DAOCS, at least in the presence of the natural substrate, penicillin N. It is possible that in the presence of an unnatural substrate, the catalytic process undergoes a more complex mechanism, possibly with the direct involvement of reducing agents in the system.

<p>Epoxides are three-membered cyclic ethers formed in the metabolism of foreign substances and as endogenous metabolites. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to yield the corresponding diols. EHs have been implicated in diverse functions such as detoxification of various toxic epoxides, as well as regulation of signal substance levels.</p><p>The main goal of this thesis was to investigate and characterize the α/β hydrolase fold EH. The first part concerns the identifictaion of an EH in <i>Saccharomyces cerevisiae</i>. The second part involves detailed mechanistic and structural studies of a plant EH from potato, StEH1. </p><p>Despite the important function of EH, no EH has previously been established in <i>S. cerevisiae</i>. By sequence analysis, we have identified a new subclass of EH present in yeast and in a wide range of microorganisms. The <i>S. cerevisiae</i> protein was produced recombinantly and was shown to display low catalytic activity with tested epoxide substrates. </p><p>In plants, EHs are involved in the general defence system, both in the metabolism of the cutin layer and in stress response to pathogens. The catalytic mechanism of recombinantly expressed wild type and mutant potato EH were investigated in detail using the two enantiomers of <i>trans</i>-stilbene oxide (TSO). The proposed catalytic residues of StEH1 were confirmed. StEH1 is slightly enantioselective for the <i>S,S</i>-enantiomer of<i> trans</i>-stilbene oxide. Furthermore, distinct pH dependence of the two enantiomers probably reflects differences in the microscopic rate constants of the substrates. The detailed function of the two catalytic tyrosines was also studied. The behavior of the tyrosine pair resembles that of a bidentate Lewis acid and we conclude that these tyrosines function as Lewis acids rather then proton donors.</p><p>The three dimensional structure of StEH1 was solved, representing the first structure of a plant EH. The structure provided information about the substrate specificity of StEH1.</p>

Epoxides are three-membered cyclic ethers formed in the metabolism of foreign substances and as endogenous metabolites. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to yield the corresponding diols. EHs have been implicated in diverse functions such as detoxification of various toxic epoxides, as well as regulation of signal substance levels. The main goal of this thesis was to investigate and characterize the α/β hydrolase fold EH. The first part concerns the identifictaion of an EH in Saccharomyces cerevisiae. The second part involves detailed mechanistic and structural studies of a plant EH from potato, StEH1. Despite the important function of EH, no EH has previously been established in S. cerevisiae. By sequence analysis, we have identified a new subclass of EH present in yeast and in a wide range of microorganisms. The S. cerevisiae protein was produced recombinantly and was shown to display low catalytic activity with tested epoxide substrates. In plants, EHs are involved in the general defence system, both in the metabolism of the cutin layer and in stress response to pathogens. The catalytic mechanism of recombinantly expressed wild type and mutant potato EH were investigated in detail using the two enantiomers of trans-stilbene oxide (TSO). The proposed catalytic residues of StEH1 were confirmed. StEH1 is slightly enantioselective for the S,S-enantiomer of trans-stilbene oxide. Furthermore, distinct pH dependence of the two enantiomers probably reflects differences in the microscopic rate constants of the substrates. The detailed function of the two catalytic tyrosines was also studied. The behavior of the tyrosine pair resembles that of a bidentate Lewis acid and we conclude that these tyrosines function as Lewis acids rather then proton donors. The three dimensional structure of StEH1 was solved, representing the first structure of a plant EH. The structure provided information about the substrate specificity of StEH1.

Enolate formation by proton abstraction from an sp3-hybridized carbon atom situated next to a carbonyl or carboxylate group is an abundant process in nature. Since the corresponding nonenzymatic process in water is slow and unfavorable due to high intrinsic free energy barriers and high substrate pKa s, enzymes catalyzing such reaction steps must overcome both kinetic and thermodynamic obstacles. Computer simulations were used to study enolate formation catalyzed by glyoxalase I (GlxI) and 3-oxo-Δ5-steroid isomerase (KSI). The results, which reproduce experimental kinetic data, indicate that for both enzymes the free energy barrier reduction originates mainly from the balancing of substrate and catalytic base pKas. This was found to be accomplished primarily by electrostatic interactions. The results also suggest that the remaining barrier reduction can be explained by the lower reorganization energy in the preorganized enzyme compared to the solution reaction. Moreover, it seems that quantum effects, arising from zero-point vibrations and proton tunnelling, do not contribute significantly to the barrier reduction in GlxI. For KSI, the formation of a low-barrier hydrogen bond between the enzyme and the enolate, which is suggested to stabilize the enolate, was investigated and found unlikely. The low pKa of the catalytic base in the nonpolar active site of KSI may possibly be explained by the presence of a water molecule not detected by experiments. The hemoglobin-degrading aspartic proteases plasmepsinI and plasmepsin II from Plasmodium falciparum have emerged as putative drug targets against malaria. A series of C2- symmetric compounds with a 1,2-dihydroxyethylene scaffold were investigated for plasmepsin affinity, using computer simulations and enzyme inhibition assays. The calculations correctly predicted the stereochemical preferences of the scaffold and the effect of chemical modifications. Calculated absolute binding free energies reproduced experimental data well. As these inhibitors have down to subnanomolar inhibition constants of the plasmepsins and no measurable affinity to human cathepsin D, they constitute promising lead compounds for further drug development.

5-aminolevulinic acid (5-ALA) and derivatives thereof are widely usedprodrugs in treatment of pre-malignant skin diseases of the cancer treatmentmethod photodynamic therapy (PDT). The target molecule in 5-ALAPDTis protoporphyrin IX (PpIX), which is synthesized endogenously from5-ALA via the heme pathway in the cell. This thesis is focused on 5-ALA,which is studied in different perspectives and with a variety of computationalmethods. The structural and energetic properties of 5-ALA, itsmethyl-, ethyl- and hexyl esters, four different 5-ALA enols, and hydrated5-ALA have been investigated using Quantum Mechanical (QM) first principlesdensity functional theory (DFT) calculations. 5-ALA is found to bemore stable than its isomers and the hydrolysations of the esters are morespontaneous for longer 5-ALA ester chains than shorter. The keto-enoltautomerization mechanism of 5-ALA has been studied, and a self-catalysismechanism has been proposed to be the most probable. Molecular Dynamics(MD) simulations of a lipid bilayer have been performed to study themembrane permeability of 5-ALA and its esters. The methyl ester of 5-ALAwas found to have the highest permeability constant (PMe-5-ALA = 52.8 cm/s).The mechanism of the two heme pathway enzymes; Porphobilinogen synthase(PBGS) and Uroporphyrinogen III decarboxylase (UROD), have beenstudied by DFT calculations and QM/MM methodology. The rate-limitingstep is found to have a barrier of 19.4 kcal/mol for PBGS and 13.7kcal/mol for the first decarboxylation step in UROD. Generally, the resultsare in good agreement with experimental results available to date.

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