The Diels-Alder reaction is one of the most powerful synthetic tools in organic chemistry, and asymmetric Diels-Alder catalysis allows for rapid construction of chiral carbon scaffolds. For this reason, considerable effort has been invested in developing efficient and stereoselective organo- and biocatalysts. However, Diels-Alder is a virtually unknown reaction in Nature, and to engineer an enzyme into a Diels-Alderase is therefore a challenging task. Despite several successful designs of catalytic antibodies since the 1980’s, their catalytic activities have remained low, and no true artificial ’Diels-Alderase’ enzyme was reported before 2010. In this thesis, we employ state-of-the-art computational tools to study the mechanism of organocatalyzed Diels-Alder in detail, and to redesign existing enzymes into intermolecular Diels-Alder catalysts. Papers I–IV explore the mechanistic variations when employing increasingly activated reactants and the effect of catalysis. In particular, the relation between the traditionally presumed concerted mechanism and a stepwise pathway, forming one bond at a time, is probed. Papers V–X deal with enzyme design and the computational aspects of predicting catalytic activity. Four novel, computationally designed Diels-Alderase candidates are presented in Papers VI–IX. In Paper X, a new parameterization of the Linear Interaction Energy model for predicting protein-ligand affinities is presented. A general finding in this thesis is that it is difficult to attain large transition state stabilization effects solely by hydrogen bond catalysis. In addition, water (the preferred solvent of enzymes) is well-known for catalyzing Diels- Alder by itself. Therefore, an efficient Diels-Alderase must rely on large binding affinities for the two substrates and preferential binding conformations close to the transition state geometry. In Papers VI–VIII, we co-designed the enzyme active site and substrates in order to achieve the best possible complementarity and maximize binding affinity and pre-organization. Even so, catalysis is limited by the maximum possible stabilization offered by hydrogen bonds, and by the inherently large energy barrier associated with the [4+2] cycloaddition. The stepwise Diels-Alder pathway, proceeding via a zwitterionic intermediate, may offer a productive alternative for enzyme catalysis, since an enzyme active site may be more differentiated towards stabilizing the high-energy states than for the standard mechanism. In Papers I and III, it is demonstrated that a hydrogen bond donor catalyst provides more stabilization of transition states having pronounced charge-transfer character, which shifts the preference towards a stepwise mechanism. Another alternative, explored in Paper IX, is to use an α,β -unsaturated ketone as a ’pro-diene’, and let the enzyme generate the diene in situ by general acid/base catalysis. The results show that the potential reduction in the reaction barrier with such a mechanism is much larger than for conventional Diels-Alder. Moreover, an acid/base-mediated pathway is a better mimic of how natural enzymes function, since remarkably few catalyze their reactions solely by non-covalent interactions. / <p>QC 20120903</p>

Iron (Fe) oxy-hydroxides such as ferrihydrite (Fh) are ubiquitous in surface environments. Because of their high surface area and high reactive surface, they can immobilize environmental contaminants and nutrients (e.g., oxyanions) through adsorption. Ferrihydrite is metastable and eventually transforms to hematite (Hm), goethite (Gt), and lepidocrocite (Lp). Although the Fh formation and transformation and oxyanion adsorption on its surface have been separately studied, the coupled interaction of these processes is only partly understood. The impact of oxyanion surface complexes on the rate and pathway of Fh transformation was studied. Results show that AsO43- and SO42- inner-sphere complexes decrease the rate of Fh transformation and induce the formation of Hm. In contrast, NO3- outer-sphere complexes promote the formation of Gt. We then investigated the impact of oxyanion (AsO43- and PO43-) surface loading on the rate and pathway of Fh transformation. The results show that the rate of Fh transformation decreases, and more Hm forms with increasing the oxyanion surface loading. Cryogenic transmission electron microscopy (Cryo-TEM) was also used to study the effect of oxyanion surface complexes (NO3- and PO43-) on the nucleation and growth of Gt and Hm during Fh transformation. Our results show that Gt first was formed from Fh dissolution and then grew by oriented attachment. In contrast, Hm formed after the aggregation of Fh particles. We propose that NO3- outer-sphere complexes hydrate the surface and promote the Gt formation through a dissolution/crystallization pathway, while PO43- inner-sphere complexes dehydrate the surface and induce more Hm through an aggregation pathway. In the final project, we investigated the formation of Fh from Fe oxy-hydroxide clusters. The results showed that increasing pH increased the size and structural order of particles that resemble 2-line Fh. Also, the particle size of aged samples at pHs 1.5 and 2.5 increased with time, and they transformed to Gt and Lp. In this work, we develop new ways to study the formation and transformation of Fh. These methods and information can be used to develop further studies towards a comprehensive understanding of Fh formation and transformation in other environmental conditions, such as redox systems. / Doctor of Philosophy / Iron (Fe) oxy-hydroxides nanoparticles are composed of Fe, oxygen (O), and water (H2O). One of the most famous Fe nanoparticles is ferrihydrite (Fh), which is commonly found in soils, sediments, and water. Ferrihydrite surface is positively charged and adsorbs negatively charged ions such as oxyanions, which are important contaminants (e.g., arsenate; AsO43- and sulfate; SO42) and nutrients (e.g., phosphate; PO43- and nitrate; NO3-) in drinking water in the US and around the world. The structure of Fh is not stable, and it transforms to other Fe oxy-hydroxides such as goethite (Gt), hematite (Hm), and lepidocrocite (Lp). Pre-adsorbed oxyanions may release during Fh transformation and impact water and soil quality. Additionally, oxyanion adsorption may affect the formation and transformation of Fh, which is not fully understood. In this work, we investigate how oxyanions change the rate and pathway of Fh transformation. The results show that the strong binding of AsO43- and SO42- slows down the rate of Fh transformation and favors the formation of Hm as opposed to Gt. While weak adsorption of NO3- promotes the formation of more Gt. We also study the transformation of Fh in the presence of different oxyanions (AsO43- and PO43-) concentrations. The results show that the rate at which Fh transforms to Gt and Hm decreases, and more Hm forms with increasing the concentration of oxyanions on the Fh surface. Interestingly, results also show that weekly bounded NO3- and SO42- could be released to the solution phase, while strongly adsorbed AsO43- and PO43- could remain on the surface during the ferrihydrite transformation. We also used an imaging technique (Cryogenic transmission electron microscopy; Cryo-TEM) to study the effect of oxyanion surface complexes (NO3- and PO43-) on the formation and growth of Gt and Hm during the Fh transformation. The results show that Gt first was formed from Fh and then grew to larger particles. In contrast, for the formation of Hm, Fh particles first aggregate, form larger particles, and then transform to Hm. In the final project, we investigate the formation of Fh from Fe oxy-hydroxide cluster precursors. Results show that Fe oxy-hydroxide clusters can be the potential precursors for forming Fh during the rapid hydrolysis of Fe(III) solutions. However, when these precursors are aged, they do not form Fh and transform to Gt and Lp, which have stable structures. In this work, we develop new ways to study the formation and transformation of Fh, which will have implications for understanding how and when contaminant remobilization will occur during Fh formation and transformation.

Yes / The crotonases comprise a widely distributed enzyme superfamily that has multiple roles in both primary and secondary metabolism. Many crotonases employ oxyanion hole-mediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) thioester substrates (e.g., malonyl-CoA, α,β-unsaturated CoA esters) both with nucleophiles and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved fold formed from a central β-sheet core surrounded by α-helices, which typically oligomerizes to form a trimer or dimer of trimers. The presence of a common structural platform and mechanisms involving intermediates with diverse reactivity implies that crotonases have considerable potential for biocatalysis and synthetic biology, as supported by pioneering protein engineering studies on them. In this Perspective, we give an overview of crotonase diversity and structural biology and then illustrate the scope of crotonase catalysis and potential for biocatalysis. / Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Wellcome Trust

This thesis describes the use of rational redesign to modify the properties of the enzyme Candida antarctica lipase B. Through carefully selected single-point mutations, we were able to introduce substrate-assisted catalysis and to alter the reaction specificity. Other single-point mutations afforded variants with greatly changed substrate selectivity and enantioselectivity. Mutation of the catalytic serine changed the hydrolase activity into an aldolase activity. The mutation decreased the activation energy for aldol addition by 4 kJ×mol-1, while the activation energy increased so much for hydrolysis that no hydrolysis activity could be detected. This mutant can catalyze aldol additions that no natural aldolases can catalyze. Mutation of the threonine in the oxyanion hole proved the great importance of its hydroxyl group in the transition-state stabilization. The lost transition-state stabilization was partly replaced through substrate-assisted catalysis with substrates carrying a hydroxyl group. The poor selectivity of the wild-type lipase for ethyl 2-hydroxypropanoate (E=1.6) was greatly improved in the mutant (E=22), since only one enantiomer could perform substrate-assisted catalysis. The redesign of the size of the stereospecificity pocket was very successful. Mutation of the tryptophan at the bottom of this pocket removed steric interactions with secondary alcohols that have to position a substituent larger than an ethyl in this pocket. This mutation increased the activity 5 500 times towards 5-nonanol and 130 000 times towards (S)-1-phenylethanol. The acceptance of such large substituents (butyl and phenyl) in the redesigned stereospecificity pocket increases the utility of lipases in biocatalysis. The improved activity with (S)-1-phenylethanol strongly contributed to the 8 300 000 times change in enantioselectivity towards 1-phenylethanol; example of such a large change was not found in the literature. The S-selectivity of the mutant is unique for lipases. Its enantioselectivity increases strongly with temperature reaching a useful S-selectivity (E=44) at 69 °C. Thermodynamics analysis of the enantioselectivity showed that the mutation in the stereospecificity pocket mainly changed the entropic term, while the enthalpic term was only slightly affected. This pinpoints the importance of entropy in enzyme catalysis and entropy should not be neglected in rational redesign.