Computational studies of mechanisms, spectra and conformations in biological systems.
PhD thesis, University of Glasgow.
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This thesis represents computational studies of the mechanisms of chlorination in the FAD-dependent enzyme PrnA using QM, MD and QM/MM methods. It also contains Time-Dependent Density Functional Theory (TDDFT) spectra and conformational analysis of the fluoro-imine derivatives of pyridoxal 5’-phosphate (PLP).
Chapter 1 gives a brief introduction of the theoretical methods used in the thesis and an explanation of their implementation in Computational Chemistry. Chapter 2 presents an overview of the variety of halogenated natural products and halogenating enzymes. This chapter takes a closer look at the group of the FAD-dependent enzymes and in particular at PrnA. All the feasible mechanisms of regioselective chlorination of tryptophan in PrnA are thoroughly discussed. In Chapter 3 model reactions for the chlorination of the substrate tryptophan at the DFT level are investigated. Two different chlorinating agents are proposed and the reaction energies and barriers for the halogenating reactions are calculated. A simple comparison of the chlorinating agents at the QM level without the enzyme is given. Chapters 4 and 5 are devoted to MD simulations on PrnA for equilibration of the solvated protein and QM/MM modelling with the AM1/AMBER03 and the DFT/AMBER03 methods. The QM/MM methods gave the opportunity of studying the reaction mechanisms with the account of the environment of the enzyme. All the steps along the mechanisms of chlorination with the possible chlorinating agents were investigated.
In chapter 6 TDDFT has been used to calculate the electronic spectra of PLP and its derivatives. PLP is co-factor in a diversity of enzymes with different biological
function. In the alanine racemase it takes part in the racemisation of L-alanine to D-alanine. The latter is a building block of the bacterial cell wall. A novel method of blocking the function of the enzyme is the use of the β,β,β-trifluoroalanine inhibitor which forms an external aldimine with PLP. A mechanism of the β,β,β-trifluoroalanine inhibitor activity was proposed by Faraci and Walsh. The spectra at all the steps along the proposed mechanism with intermediates in different protonation states were calculated.
Chapter 7 focusses on the examination of the conformations of the PLP derivatives and the energetics of these compounds. The preference of gauche conformation in β-
fluoroimines was studied. In this chapter are presented empirical (X-ray) and theoretical (DFT) evidence for the gauche effect in β-fluoroimines. NCCF bond rotational profiles of acetaldehyde-derived imines and β-fluoropyridoximine at the DFT level were analysed. NBO analysis of the investigated compounds proved the Dunathan’s stereoelectronic hypothesis.
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