The Fdc1 and Pad1 decarboxylase system from Saccharomyces cerevisiae has been identified as a potential candidate to feature in novel biofuel production pathways based on its ability to catalyze the transformation of sorbic acid, an antimicrobial compound, to 1, 3-pentadiene, a volatile hydrocarbon. Although information on the system is currently insufficient to permit a full assessment of its potential for future commercialization, it is hoped that (rational) engineering approaches can be used to evolve the enzymes to produce more desirable hydrocarbons. This requires biochemical characterization of the proteins. Genetic manipulation experiments have indicated that both enzymes are required for activity. However, no in vitro studies were conducted to verify the function, determine the relationship or establish the cofactor requirements of Fdc1 and Pad1. Results reported here establish that Fdc1 is the enzyme responsible for catalyzing decarboxylation, requiring a novel cofactor synthesized by Pad1 (or the bacterial homologue UbiX) for activity. High resolution crystal structures and mass spectrometry data from Fdc1 co-expressed with UbiX have indicated that the cofactor corresponds to a modified flavin mononucleotide (FMN) that has been extended with a C5-unit through linkages at the N5 and C6 atoms, creating a fourth, non-aromatic ring on the isoalloxazine group. Subsequent solution studies have established that this modification is achieved through isoprene chemistry, with UbiX facilitating prenyl transfer from dimethylallyl monophosphate (DMAP) to FMN. Analysis of wild type and mutant UbiX constructs by kinetic X-ray crystallography has allowed several distinct stages of the prenyl transfer reaction to be trapped, establishing that the protein uses a number of chemical strategies similar to terpene synthases to generate its product. The active site is dominated by pi systems, which aid heterolytic cleavage of the isoprene precursors phosphate-C1’ bond following FMN reduction, leading to the formation of an N5-C1’ intermediate. UbiX then acts as a chaperone for adduct reorientation, potentially via a transient tertiary carbocation, ultimately resulting in ring closure between the C6 and C3’. This work has established the biochemical principles underpinning the Fdc1 and Pad/UbiX decarboxylase system, providing a platform from which rational evolution approaches can be applied to the enzymes, specifically Fdc1, to improve their validity in the biofuels industry. It has also identified a novel cofactor that extends the previously well-documented flavin and isoprenoid repertoire.

The kinetics of the thermal decarboxylation of a series of substituted sodium phenylpropiolates were studied in detail. The reaction proceeded without formation of significant amounts of side products: its rate was independent of hydrogen ion concentration. The relative rates of decarboxylation are in the order: p-NO2 > m-NO2> m-CF3 > m-Cl > p-Cl > H > m-CH3, and the entropies of activation are large and positive. The results were discussed in terms of the unimolecular (SE1) mechanism. A quantitative study of effects of substituents on the decarboxylation rates was made by the application of the Hammett free energy relationship. The e value of the Hammett plot was 0.886 +/- 0.013. Deviation of para substituted compounds from the Hammett relationship were discussed in terms of para interaction or conjugation effects in the initial or the transition states. / Thesis / Master of Science (MS)

The major goals of this thesis were to (1) design and synthesize a supported catalyst with well-defined monodisperse palladium nanoparticles evenly distributed throughout an inorganic oxide substrate with tunable porosity characteristics, (2) demonstrate the catalytic activity of this material in the decarboxylation of long chain fatty acids and their derivatives to make diesel-length hydrocarbons, (3) elucidate the deactivation mechanism of supported palladium catalysts under decarboxylation conditions via post mortem catalyst characterization and develop a regeneration methodology thereupon, and (4) apply this catalytic system to a real low-value biofeedstock. In an effort to maximize loading and minimize mass transfer limitations, mesoporous silica MCF was synthesized as catalyst support. Functionalization with various silane ligands facilitated even distribution of palladium precursor salts throughout the catalyst particle, and, after reduction, monodisperse palladium nanoparticles approximately 2 nm in diameter. The Pd-MCF catalyst showed high one-time activity in the decarboxylation of fatty acids to hydrocarbons in dodecane at 300 °C. Subsequent reactions were performed on acid derivatives to elucidate a decarboxylation reaction pathway. The catalyst experienced severe deactivation after only one use and substantial effort was put into elucidating the nature of this deactivation via post mortem catalyst characterization. The deactivation was found not to be caused by nanoparticle sintering, agglomeration or ripening, but instead by organic deposition, mainly of reactant acid. A regeneration methodology was developed and subsequent catalyst reuse exhibited high activity. Finally, the Pd-MCF catalyst was applied to a wastewater-derived brown grease from a poultry rendering facility, in an unpolished and polished form. The latter was successfully decarboxylated to diesel-length hydrocarbons with high conversion and selectivity.

Stopped-flow experiments in which the NAD-malic enzyme was preincubated with different reactants at near saturating substrate concentrations suggest a slow isomerization of the E:NAD:Mg complex. The lag is eliminated by preincubation with Mg˙² and malate suggesting that the formation of E:Mg:Malate either bypasses or speeds up the slow isomerization step. Circular dichroic spectral studies of the secondary structural changes of the native enzyme in the presence and absence of substrates supports the existence of conformational changes with NAD˙ and malate. Thus, a slow conformational change of the E:NAD:Mg complex is likely one of the rate-limiting steps in the pre-steady state.

Sphingolipids and ceramides are essential components of cellular membranes and important signalling molecules. Because of a growing appreciation for their diverse biological roles, understanding of the biosynthesis and regulation of sphingolipids has recently become a key goal in drug discovery. Serine palmitoyltransferase (SPT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyses the condensation between L-serine and a long-chain acyl thioester such as palmitoyl-CoA (C16-CoA). This first step in sphingolipid biosynthesis is conserved in all organisms studied to date, from microbes to man. The fungal natural product myriocin is a potent inhibitor of SPT; however, the molecular details of inhibition are not fully understood. Myriocin contains a long alkyl chain and a polar head group thus it displays features of both SPT substrates. Therefore, the prevailing hypothesis is that inhibition of SPT occurs because myriocin acts as a mimic of a key transition state of the catalytic mechanism. Through a combination of UV-vis spectroscopy, mass spectrometry, x-ray crystallography and enzyme inhibition assays it has been possible to study the interaction between S. paucimobilis SPT and myriocin. I have shown that myriocin initially forms an inhibitory PLP:myriocin aldimine complex in the active site that displays a Ki of 967 nM. Interestingly, this complex is susceptible to unexpected, slow enzymatic degradation. The mechanism for myriocin breakdown has been elucidated as a retro-aldol type reaction, which results in cleavage of the C2-C3 bond producing a C18 aldehyde. This aldehyde is then capable of covalently modifying the active site lysine265, forming a second (suicide) inhibitory complex and rendering the enzyme catalytically inactive. Substitution of the active site lysine produced SPT K265A, an inactive enzyme that did not catalyse the breakdown of the PLP:myriocin complex. However, the determination of the crystal structure of the SPT K265A:PLP-myriocin complex revealed that the myriocin had undergone decarboxylation. Nevertheless, this SPT:PLP:decarboxymyriocin structure revealed details about myriocin’s mechanism of inhibition for the first time. The novel mechanism of myriocin degradation has implications on the structure activity relationship (SAR) and design of drugs targeted towards SPT, the role of feedback regulation by long chain aldehydes and further expands the range of reactions catalysed by this important enzyme. As well as inhibition studies the structure of bacterial SPT was also examined by preparing an N-terminally truncated S. paucimobilis SPT. This version, shortened by 21 amino acids, was ~5-fold slower than the wild-type enzyme and suggests that the N-terminus may play a role in catalysis. Additional work has been undertaken to study an unusual membrane-bound viral SPT, composed of two naturally fused open reading frames (SPT2-SPT1) with the proposed SPT2 domain at the N-terminus and the SPT1 domain at the C-terminus. To study soluble mimics of this interesting fusion I prepared a bacterial S. paucimobilis SPT fused wild-type and mutant construct and isolated a fused SPT2-SPT1 with what appears to be single PLPbinding site.

Some interesting, biologically active natural products have been found to contain poly-azole fragments within their core. These fragments are linked through the 2- position of one azole and the 4-position of its neighbour. Decarboxylative cross-coupling methodology may provide a route to these desired linked azoles through cross-coupling of azole-4-carboxylic acids with azole-2- halides or with azoles containing no substitution at the 2-position. Investigations into the silver-mediated decarboxylation, and subsequent coupling potential, of thiazole and oxazole-4-carboxylic acids are reported. Methods towards the synthesis of novel chlorinated thiazole and oxazole acids and their precursors are also described. A method to successfully couple these acids to aryl iodides has been developed and the scope of this reaction extended to a variety of functionalised azole-4-carboxylic acids. Attempts to extend this methodology and combine the decarboxylative coupling with CH activation of a second azole are also described.

Density Functional Theory (DFT) is a powerful tool for treating large organometallic structures efficiently and accurately. DFT calculations on the Hg-catalyzed oxidation of methane to methyl bisulfate in sulfuric acid suggest the lowest energy pathway involves a closed-shell electrophilic C–H activation mechanism coupled with metal alkyl reductive functionalization and oxidation by SO3. Comparison to Tl, Zn, and Cd suggests that Hg is unique in its ability to catalyze this set of reaction steps. Comparison to K2S2O8 highlights the selectivity of this C–H activation reaction as opposed to radical conditions. In contrast, DFT calculations indicate that CoIII(TFA)3 oxidizes methane through a radical TFA ligand decarboxylation pathway. A similar decarboxylation pathway is identified for MnIII(TFA)3, but the low spin ground state of TlIII(TFA)3 favors electrophilic C–H activation over this decarboxylation pathway. DFT calculations indicate that Cp(PPh2Me)Co=CF2 undergoes [2 + 2] cycloaddition with TFE by a unique open-shell singlet diradical mechanism. The significant stability of the perfluorometallacyclobutane reveals why catalytic metathesis with TFE is difficult.

Diminishing petroleum reserves and environmental considerations have strengthened the demand for developing renewable fuel technologies. One alternative is deoxygenating plant oils, animal fats, and waste lipid streams to fuel-like hydrocarbons. These fuels offer a drop-in replacement to petroleum products while potentially becoming carbon neutral, satisfying both fuel and environmental concerns. This fuel is obtained through catalytic deoxygenation via either hydrodeoxygenation (HDO) or decarboxylation/ decarbonylation (deCOx). HDO requires problematic sulfided catalysts and extreme hydrogen pressures to convert lipids to fuel-like hydrocarbons. Therefore, this work focuses on the deCOx pathway, where hydrogen is not required for deoxygenation to take place. Generally, other authors use Pd or Pt as the active metals for deCOx; however, their cost can be industrially prohibitive. Recently, inexpensive Ni catalysts have shown comparable catalytic deCOx activity to Pd and Pt, albeit significant catalyst deactivation and catalytic cracking to undesirable products remain problematic. Therefore, this work aims to improve the activity, selectivity, and recyclability of supported Ni catalysts for the deCOx of lipids. Cu, Sn, and minimal amount of Pt were investigated as secondary promoter metals for Ni catalysts for deCOx. Deoxygenation of waste lipids such as brown grease and yellow grease was also accomplished in an industrially relevant fixed bed reactor.

Computational methods are very useful tools in the study of enzymatic reactions, as they can provide a detailed understanding of reaction mechanisms and the sources of various selectivities. In this thesis, density functional theory has been employed to examine four different enzymes of potential importance for biocatalytic applications. The enzymes considered are limonene epoxide hydrolase, soluble epoxide hydrolase, arylmalonate decarboxylase and phenolic acid decarboxylase. Besides the reaction mechanisms, the enantioselectivities in three of these enzymes have also been investigated in detail. In all studies, quite large quantum chemical cluster models of the active sites have been used. In particular, the models have to account for the chiral environment of the active site in order to reproduce and rationalize the experimentally observed selectivities. For both epoxide hydrolases, the calculated enantioselectivities are in good agreement with experiments. In addition, explanations for the change in stereochemical outcome for the mutants of limonene epoxide hydrolase, and for the observed enantioconvergency in the soluble epoxide hydrolase are presented. The reaction mechanisms of the two decarboxylases are found to involve the formation of an enediolate- or a quinone methide intermediate, supporting thus the main features of the proposed mechanisms in both cases. For arylmalonate decarboxylase, an explanation for the observed enantioselectivity is also presented. In addition to the obtained chemical insights, the results presented in this thesis demonstrate that the quantum chemical cluster approach is indeed a valuable tool in the field of asymmetric biocatalysis. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p><p> </p>

The catechol meta-fission pathway, a degradation pathway for simple aromatic compounds, is rich in enzyme chemistry and replete with structural and evolutionary diversity. Vinyl pyruvate hydratase (VPH) and MhpD catalyze the same reaction in this pathway, but in different bacterial species. These metal ion-dependent enzymes reportedly catalyze a 1,5-keto-enol tautomerization reaction followed by a Michael addition of water. MhpD, and most likely VPH, are members of the fumarylacetoacetate hydrolase (FAH) superfamily. The crystal structure of MhpD and the sequence of VPH identified four potential active site residues, Lys-60, Leu-72, Asp-78, and Ser-160 (Ser-161 in VPH). The K60A and D78N mutants of VPH and MhpD had the most damaging effects on catalysis. Moreover, the K60A mutant seemingly uncoupled tautomerization from hydration and provided evidence for an [alpha, beta]-unsaturated ketone in the reaction. The effects of the L72A and S160A (S161A in VPH) mutants were smaller, suggesting less important roles in the mechanism. 5-(carboxymethyl)-2-Oxo-3-hexene-1,6-dioate decarboxylase (COHED) is a metal ion-dependent enzyme in the homoprotocatechuate (HPC) pathway, a chromosomally encoded meta-fission pathway from Escherichia coli C that parallels the catechol meta-fission pathway. COHED is also a member of the FAH superfamily. It is a monomeric protein with two domains. It is postulated that the C-terminal domain catalyzes the decarboxylation reaction and the N-terminal domain carries out the 1,3-keto-enol tautomerization reaction. Site-directed mutagenesis, NMR, and kinetic analysis with different substrates and inhibitors have identified three potential active-site residues Glu-276, Glu-278 (in the C-terminal domain), and Lys-110 (in the N-terminal domain). Replacement of either glutamate with a glutamine eliminated both the decarboxylase and tautomerase activities. The K110A mutant also diminished both activities, but more importantly eliminated the C-3 proton/deuteron exchange reaction observed for substrate analogs. The enzymes of the catechol and homoprotocatechuate pathways provide examples of enzyme optimization toward a specific substrate even among related compounds, as reflected by the FAH superfamily. Hence, the results of these studies add to the growing body of information about how enzymes evolve and how pathways are assembled. / text