Enzyme design is a steadily growing field of computational chemistry, but its successes are limited by the current available knowledge and application of enzyme conformational dynamics. In this work a series of FTIR and 2D IR spectroscopic methods, for observing the conformational dynamics of an enzymatic active site and its surrounding residues, are characterized. The enzyme model system for these studies is the promiscuous ene-reductase from Pyrococcus horikoshii (PhENR) which is capable of binding substrates in multiple orientations. In one method, the spectral lineshape of an aryl-nitrile substrate-analog vibrational label is analyzed using a frequency fluctuation correlation function (FFCF) and compared to the lineshape of a corresponding aryl-azide label. This analysis revealed dynamic and electrostatic active site anisotropy which may influence substrate catalysis. The second method utilizes the intramolecular vibrations of the enzymatic cofactor, flavin mononucleotide (FMN), which is shown to be sensitive to electric field changes associated with substrate binding. The final method places a site-specific nonnatural amino acid containing an azide probe within the enzyme’s hydrophobic core. Additionally, a double-mutant cycle was identified via a common design program, the Rosetta Modeling Suite, and used to analyze the effects of mutation on enzyme dynamics. Altogether, these methods demonstrate the ability of 2D IR spectroscopy to observe enzyme conformational dynamics. Application of these methods to various other enzyme model systems should provide valuable insight for the improvement of future dynamic enzyme design protocols.

The creation of artificial enzymes to catalyze desired reactions is a major goal of computational protein design. However, de novo enzymes display low catalytic efficiencies, requiring the introduction of activity-enhancing active site and distal mutations through directed evolution. A better understanding of how mutations introduced by directed evolution contribute to increased enzymatic activity will guide the development of design methods such that efficient enzymes can be designed de novo. Here, we evaluate the structural, functional, and dynamical impacts of active site and distal mutations introduced by directed evolution of the de novo retro-aldolase RA95, an enzyme that presents an important case study in enzyme design due to the significant structural remodelling that was observed during evolution. We observe that the variant RA95-Core, containing only active site mutations introduced by directed evolution, displays activity within one order of magnitude of the fully evolved variant. This suggests that computational enzyme design methods can be improved to create much more efficient enzymes than what was previously achieved in RA95. However, structural changes induced by distal mutations prevent computational recapitulation of the evolved active site on the original design template, indicating that the optimized active site identified through directed evolution could not have been designed de novo using current design methodologies. We suggest strategies for the incorporation of backbone remodelling into design procedures that would allow recapitulation of the evolved retro-aldolase active site, as well as the de novo design of highly efficient enzymes without the need for optimization by directed evolution.

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>

This thesis has been focused around the Diels Alder reaction with the goal to design an enzyme catalyzed reaction pathway. To achieve this goal computer aided enzyme design was utilized. Common traditional methods of computational chemistry (B3LYP, MP2) do not do well when calculating reaction barriers or even reaction energies for the Diels Alder reaction. New calcu- lation methods were developed and tested. This was the focus of the first part of the thesis, by choosing a small system, extensive and heavy calculations could be done with CBS-QB3. Then by benchmarking faster methods of calculation (SCS-MP2, M06-2X) against the results, they could be graded by efficiency and cost. This was done anticipating that the same accuracy could be applied to larger systems where CBS-QB3 cannot be used. In the second part activating groups were investigated for both the diene and the dienophile, along with their effects on reaction rates. A qualitative analysis was done. This is important not only for the uncatalyzed reaction, but also interesting when searching for possible substrates for the enzyme reaction. In the last part the thesis presents a designed enzyme that catalyzes Diels Alder in silico using ∆5−3−Keto steroid isomerase. Using empirical calculations, the enzyme was scanned for catalytic activity. The catalytic effect was then showed with ab initio Quantum chemical calculations.

Indications that existing parameter sets of extended Linear Interaction Energy (LIE) models are transferable between lipases from Rhizomucor Miehei and Thermomyces Lanigunosus in complex with a small set of vinyl esters are demonstrated. By calculat- ing energy terms that represents the cost of forming cavities filled by the ligand and the complex we can add them to a LIE model with en established parameter set. The levels of precision attained will be comparable to those of an optimal fit. It is also demonstrated that the Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) methods are in- applicable to the problem of calculating absolute binding energies, even when the largest source of variance has been reduced.

Structural biology uncovers life's secrets by studying protein structures via techniques like X-ray crystallography. This knowledge drives advancements in protein engineering for the improvement of human lives. Yet, obtaining high-quality crystals in X-ray crystallography is challenging. To overcome this, we used Translocation ETS Leukemia protein Sterile Alpha Motif domain (TELSAM), a promising polymer-forming crystallization chaperone (PFCC), to enhance protein crystallization. Human thirty-eight-negative kinase-1 (TNK1), a key player in cancer progression, possess a ubiquitin association (UBA) domain that binds polyubiquitin and regulates TNK1 activity and stability. Although sequence analysis hints at an unconventional TNK1 UBA domain architecture, its molecular structure lacks experimental validation. To gain insight into TNK1 regulation, we fused the UBA domain to the 1TEL crystallization chaperone and obtained crystals diffracting as far as 1.53 Ã…. 1TEL enabled solution of the X-ray phases. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its 1TEL polymer and to crystallize at protein concentrations as low as 0.1 mg/mL. Our findings support a TELSAM fusion crystallization mechanism, highlighting fewer crystal contacts compared to traditional crystals. Both modeling and experimental validation indicate that the UBA domain exhibits selectivity towards polyubiquitin chain length and linkages. Radical S-adenosylmethionine (SAM) enzymes catalyze various radical-mediated substrate transformations. Despite the growing interest of computational enzyme design in industrial small molecule synthesis, radical SAM enzymes remain relatively unexplored. We used PyRosetta to leverage hydrogen bonding design (hbDes) and hydrophobic interaction design (hpDes) to enable a radical cyclization reaction on our selected substrate. Although the purified enzymes demonstrated activation potential with a reducing agent, enzymatic assays failed to exhibit activity against the reactant. To obtain successful results, addressing additional questions and issues is required, which may involve the implementation of machine learning.

The Diels-Alder is an important reaction that is one of the primary tools for synthesizing cyclic carbon structures, while simultaneously introducing up to four stereocenters in the resulting product. Not only is it a widely explored reaction in organic chemistry, but a vital tool in industry to construct novel compounds for pharmacological applications. Still, a remaining concern is the fact that upon the introduction of stereogenic carbons, the possibility of stereoselective control is greatly diminished. A common solution to the problem of undesirable stereoisomers is to employ chiral auxiliaries and ligands as means to increase the yield of a certain stereoisomer. However, incorporating these types of compounds in order to obtain an enantiomerically pure product increases the amount of synthetic steps to be regulated, implying that one or more purification steps are necessary to obtain the desired result. An accompanying thought leans toward the environmental aspect, as the principles of green chemistry are of great importance. This thesis presents the attempts to explore the possibility of engineering an enzyme that can catalyze an asymmetric Diels-Alder reaction through the use of molecular modeling. Based on previous work, the catalytically proficient enzyme ketosteroid isomerase had been deemed a probable candidate as a Diels-Alderase. To evaluate the enzyme thoroughly, a set of compounds was scored against the active binding site where the best hits against the wild type were saved and evaluated repeatedly after the introduction of rational mutations. Although no conclusive indication of an optimal design could be obtained at the end of this work, valuable insight was retrieved on plausible design strategies, which eventually could help lead to the first catalytically proficient Diels-Alderase. / Diels-Alder är en viktig reaktion då den är ett redskap för att syntetisera cykliska kolstrukturer, samtidigt som uppemot fyra stereocentra introduceras i den resulterande produkten. Reaktionen används inte enbart inom organisk kemi, utan är även ett viktigt redskap inom industriella sammanhang för att ta fram nya preparat som direkt kan tillämpas inom farmakologi. En återstående problematik är faktumet att introduktionen av nya stereogena kol bidrar till att drastiskt minska möjligheten att bibehålla en stereoselektiv kontroll. En vanlig lösning för att undvika oönskade stereoisomerer är att nyttja kirala hjälpmolekyler och ligander för att öka utbytet av en specifik stereoisomer. Dock innebär införandet av dessa hjälpmolekyler i strävan att erhålla en enantiomeriskt ren produkt ett ökat antal syntes-steg att hantera, vilket antyder att ett eller flera reningssteg är nödvändiga för att uppnå önskat resultat. Ur en miljösynpunkt är detta värt att ha i åtanke, då principerna för grön kemi är viktiga. Detta arbete utforskar möjligheterna att konstruera ett enzym som kan katalysera en asymmetrisk Diels-Alder-reaktion, med hjälp av molekylär modellering. Baserat på tidigare arbeten har enzymet ketosteroid isomeras valts ut som en potential kandidat till ett Diels-Alderase. För att noggrant evaluera enzymet så screenades ett set av substrat mot dess aktiva säte, där de bästa träffarna gentemot vildtypen sparades och återevaluerades allteftersom rationella mutationer kontinuerligt introducerades. Trots avsaknaden av klara indikationer på att en optimal design har kunnat tas fram vid slutet av detta arbete, så erhölls värdefull insikt på möjliga design-strategier, vilket skulle kunna bistå sökandet av det första katalytiskt effektiva Diels-Alderase.

Biocatalysis represents an attractive route to the production of various compounds which are difficult or impossible to synthesise and isolate using traditional chemical synthesis. In particular, the production of chiral molecules is a function ideally suited to biocatalysis, due to the natural stereospecificity of enzymes. The synthesis of such chiral molecules is essential in the production of pharmaceuticals, additives for the food and drinks industry and the creation of specialist polymers. CYP105AS1, isolated from Amycolatopsis orientalis, is a cytochrome P450 enzyme which produces the inactive 6-epi-pravastatin of the blockbuster anti-cholesterol drug pravastatin. Previous directed evolution efforts have engineered this enzyme to produce a five-point mutant, known as P450prava, which partially reversed the stereospecificity of the enzyme to produce a majority pravastatin product mixture. This thesis details work to use structure-led engineering approaches to redesign the active site of P450prava to introduce stringent stereospecificity. A combinatorial approach of manual and computational rational design was pursued, leading to the creation of a novel T95F/V180M double mutant of P450prava. This double mutant was found to have successfully eliminated the unwanted 6-epi-pravastatin enantiomer from the product mix, leaving a pure pravastatin product. P450prava was also shown to bind and hydroxylate other statin substrate molecules, demonstrating its versatility in the production of drug metabolites and other high-value oxyfunctionalised molecules. This property, along with its proven tolerance of significant active site engineering efforts, demonstrates the viability of the P450prava as a platform for the creation of novel biocatalysts for the production of various hydroxylated products from diverse substrate molecules.