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Abstract

The main goal of this work was to apply combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate two different enzymatic reactions and to develop new QM/MM techniques in the field of reaction path and free energy calculations.
Cyclohexanone Monooxygenase. The main target of the current work was the reaction mechanism of Cyclohexanone Monooxygenase (CHMO) - a representative Baeyer-Villiger monooxygenase enzyme, responsible for oxygenation of cycohexanone into ε-caprolactone in nature, but also famous for accepting and being enantioselective towards a wide range of substrates. We have for the first time modeled the cyclohexanone oxygenation reaction in the active site of CHMO, and explained the experimentally observed regio- and stereoselectivity of this enzyme towards several substituted substrates.
Lipopolysaccharyl-α-1,4 galactosyltransferase C. A QM/MM study of the reaction mechanism of lipopolysaccharyl-α-1,4 galactosyltransferase C (LgtC), a member of the family of retaining galactosyltransferase (GT) enzymes, was performed. We have assessed the different proposed mechanisms and showed that the SNi mechanism, via a front-side attack of the acceptor oxygen to the donor anomeric carbon atom, most probably takes place in the wild type enzyme. We have also analysed key interactions that help this attack by stabilizing the corresponding oxocarbenium ion-like transtion state.
Microiterative IRC. We have implemented and tested a microiterative method to perform intrinsic reaction coordinate (IRC) calculations for QM/MM systems of arbitrary size and complexity. In this approach, the reactive system is divided into a core and an outer region, with each step of the core region along the IRC being followed by the minimization of the outer region. We
have tested the microiterative IRC method for a number of systems with varying size and complexity and showed that it is an efficient and relatively accurate procedure to perform reaction path calculations for large systems.
DH-FEP. We have developed a new QM/MM free energy technique called dual Hamiltonian free energy perturbation (DH-FEP). The method is superior to the conventional QM/MM-FE scheme due to the explicit sampling of the QM region. In order to allow for sufficient sampling of the reactive system, simulations are performed at a semiempirical QM/MM level, while
the perturbation energies are evaluated at a higher level of theory (QM = DFT or MP2). The performance of the method strongly depends on the geometrical correspondence between the two levels. We have performed test calculations and suggested suitable strategies to identify the best matching pair of methods. We have also proposed the use of collective reaction coordinates within the DH-FEP method, which makes the problem of configurational space overlap between the two levels less relevant at the price of a slightly reduced sampling of the QM region.