In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which was further shown to be substrate dependent (Paper V). A homology structure of Cv-ωTA guided the creation of an enzyme variant showing reduced isopropyl amine inhibition. / QC20100609

Amine transaminases are enzymes that catalyze the mild and selective formation of primary amines, which are useful building blocks for biologically active compounds and natural products. In order to make the production of these kinds of compounds more efficient from both a practical and an environmental point of view, amine transaminases were incorporated into multi-step one-pot reactions. With this kind of methodology there is no need for isolation of intermediates, and thus unnecessary work-up steps can be omitted and formation of waste is prevented. Amine transaminases were successfully combined with other enzymes for multi-step synthesis of valuable products: With ketoreductases all four diastereomers of a 1,3-amino alcohol could be obtained, and the use of a lipase allowed for the synthesis of natural products in the form of capsaicinoids. Amine transaminases were also successfully combined with metal catalysts based on palladium or copper. This methodology allowed for the amination of alcohols and the synthesis of chiral amines such as the pharmaceutical compound Rivastigmine. These examples show that the use of amine transaminases in multi-step one-pot reactions is possible, and hopefully this concept can be further developed and applied to make industrial processes more sustainable and efficient in the future. / <p>QC 20170113</p>

Computer modeling of enzymes is a valuable complement to experiments. Quantum chemical studies of enzymatic reactions can provide a detailed description of the reaction mechanism and elucidate the roles of various residues in the active site. Different reaction pathways can be analyzed, and their feasibility be established based on calculated energy barriers. In the present thesis, density functional theory has been used to study the active sites and reaction mechanisms of three different enzymes, cytosine deaminase (CDA) from Escherichia coli, ω-transaminase from Chromobacterium violaceum (Cv-ωTA) and dinitrogenase reductase-activating glycohydrolase (DraG) from Rhodospirillum rubrum. The cluster approach has been employed to design models of the active sites based on available crystal structures. The geometries and energies of transition states and intermediates along various reaction pathways have been calculated, and used to construct the energy graphs of the reactions. In the study of CDA (Paper I), two different tautomers of a histidine residue were considered. The obtained reaction mechanism was found to support the main features of the previously proposed mechanism. The sequence of the events was established, and the residues needed for the proton transfer steps were elucidated. In the study of Cv-ωTA (Paper II and Paper III), two active site models were employed to study the conversion of two different substrates, a hydrophobic amine and an amino acid. Differences and similarities in the reaction mechanisms of the two substrates were established, and the role of an arginine residue in the dual substrate recognition was confirmed. In the study of DraG (Paper IV), two different substrate-binding modes and two different protonation states of an aspartate residue were considered. The coordination of the first-shell ligands and the substrate to the two manganese ions in the active site was characterized, and a possible proton donor in the first step of the proposed reaction mechanism was identified. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>